A massive £1.1bn investment means more people than ever are set to visit Dundee.
The city by the Tay is undergoing an incredible transformation as it becomes one of the top visitor destinations in Scotland.
Just 60 miles from Edinburgh and 80 miles from Glasgow, Dundee is buzzing with brilliant bars and culture, and fabulous food.
Much of the city's redevelopment has focused on the waterfront area – on the doorstep of the fantastic Apex City Quay Hotel & Spa.
Stepping out to the flagship development there is a definite feeling of excitement as the city responds to the pulse of progress.
At the heart of it all is the V&A Museum Dundee.
The £80m V&A Dundee project opens on September 15, when it will become Scotland's first ever dedicated design museum and the only V&A museum outside London.
The stunning V&A Dundee building is sure to be one of the top visitor attractions in the country.
The V&A Dundee is the first in the UK to be designed by renowned award-winning Japanese architects Kengo Kuma & Associates.
Those keen to see it in all its glory researching "Victoria and Albert Museum Dundee" should look no further than the Apex City Quay Hotel & Spa.
For a family trip to the V&A Dundee or a relaxing weekend break with some culture thrown in we have you covered.
The Apex hotel has complimentary high-speed Wi-Fi and a range of rooms offering spectacular views over the River Tay or the quayside, with some offering sights of the V&A Dundee.
The stylish Metro Bar and Brasserie has head Chef Graham Paulley – formerly of Gleneagles and the Old Course at St Andrews – at its helm and offers a range of dishes which pay homage to Scotland's rich culinary tradition while drawing inspiration from around the world.
As the V&A Dundee was designed by a Japanese architect we have also drawn inspiration from the east with our award winning Yu spa.
Led by the ancient Japanese belief in the healing power of hot water the Yu Spa offers more than 50 relaxing, detoxifying and rejuvenating Elemis treatments.
Once guests have enjoyed their choice of treatment, they can dive into the spa's Ozone swimming pool, Japanese-style hot tub, sauna and herbal-infused steam room. Those looking to get moving can do so in the hotel's Technogym™ studio.


With more than 200 events the Stuttgart Festival of Animated Film (ITFS) offers an enormous amount of interesting and important programs for you as an industry professional. Together with our cooperation partners, we offer you a unique platform: at the same time as ITFS, the leading industry events Animation Production Days and FMX – International Conference on Animation, Effects, Games and Immersive Media take place.
Take advantage of all benefits of an ITFS accreditation for professionals and be part of it when Stuttgart becomes the center for animation, visual effects, virtual reality and moving image communication from April 30 to May 05, 2019.
Please click here, to get to the accreditation.


I've heard a lot of support for independent book stores through Stamford Notes, and ok, this is not within the strict boundary lines of our fine city, but if you head down the road just a click, you'll come across Barrett Bookstore in Darien.
-Christina Baker Kline ("Bird in Hand").
I love Barrett Bookstore. I just bought the wife the Sookie Stackhouse box set (it's the series on which True Blood is based).


This is a two-volume set that comes with a slipcase. It's probably the best reference on Salvador Dali and his works of surrealism.
The books weigh 13 pounds so if you're getting from Amazon, there's going to be additional weight surcharge.
Picasso called Dalí "an outboard motor that's always running." Dalí thought himself a genius with a right to indulge in whatever lunacy popped into his head. Painter, sculptor, writer, and filmmaker, Salvador Dalí (1904-1989) was one of the century's greatest exhibitionists and eccentrics—and was rewarded with fierce controversy wherever he went. He was one of the first to apply the insights of Sigmund Freud and psychoanalysis to the art of painting, approaching the subconscious with extraordinary sensitivity and imagination. This lively monograph presents the infamous Surrealist in full color and in his own words. His provocative imagery is all here, from the soft watches to the notorious burning giraffe. A friend of the artist for over thirty years, privy to the reality behind Dalí`s public image, author Robert Descharnes is uniquely qualified to analyze Dalí—both the man and the myth.


text:
português (Portugese)
JoVE Journal Biology
Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.
A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor α) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.
In vitro lung cell cultures offer cost-effective, robust, and well-controlled platforms to assess the hazards of aerosols1. As a model cell system for human alveolar pneumocytes, the epithelial A549 cell line isolated from a pulmonary adenocarcinoma is often used2. These cells represent squamous type II epithelial cells from the alveolar region3 and are a widely used lung cell line for hazard and toxicity assessment1,4,5,6,7,8,9,10. The A549 cell line possesses relevant features of alveolar epithelial type II cells, such as the presence of characteristic lamellar bodies containing densely packed phospholipids3.
It has been shown that when the cells are cultured at an air-liquid interface (ALI), surfactant is released on the apical side of air-exposed epithelial cells, reducing surface tension11,12,13. This feature is particularly important in nanomaterial respiratory hazard and toxicity investigations. Once inhaled nanomaterials/toxicants are deposited in the alveolar region, they first interact with pulmonary surfactant and are displaced by wetting forces into the aqueous hypophase, where the interaction with pulmonary cells takes place14,15. Even though A549 cells form a monolayer (which can overgrow into multilayers at later timepoints when cultivated at ALI) and produce surfactant, a drawback is their insufficient tight junction formation, resulting in low transepithelial electrical resistance values, but still presenting a functional barrier against intercellular (nano)particles translocation16,17,18.
In the lungs, there is a variety of immune cell populations, including phagocytic and professional antigen-presenting cells (i.e., macrophages and dendritic cells) that directly communicate through cell-cell contact or intercellular signaling to control and maintain homeostasis. Macrophages and dendritic cells are critical innate immune effectors and initiators of the adaptive immune response19. Dendritic cells residing within or beneath the epithelium can form protrusions across the epithelium to the lumen to catch antigens. Alveolar macrophages are located on the apical surface of the epithelium and act as sentinel cells, representing the first cellular defense against foreign material as well as bacterial, viral and fungal infections. Their phenotypic plasticity allows rapidly induction of proinflammatory reactions in response to such stimuli as well as shifting into triggering anti-inflammatory (i.e., inhibitory) reactions20.
To simulate the human alveolar epithelial tissue barrier, we established a triple coculture model with A549 cells supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) on the apical and basal sides, respectively17. Cultivation of this model at ALI has been previously reported16, even up to 72 h post-exposure21. Acute immune responses to carbon nanotubes exposures was significantly enhanced in cell culture exposed to the ALI as compared to submerged conditions22. The coculture model, cultured and exposed to different materials at ALI, has been previously used to investigate cytotoxicity, oxidative stress and inflammatory responses upon exposures to zinc oxide,23 graphene-related materials24, gold nanoparticles25,26, carbon nanotubes21, and volcanic ash and diesel exhaust particles27.
Furthermore, the important role of macrophages and dendritic cells as immune effector cells in an in vitro human lung model has been confirmed. In particular, an increased proinflammatory response in the model was observed only in the presence of immune cells in comparison to monoculture systems7. Potential drawbacks of using primary monocyte-derived immune cells are the limited accessibility of PBMs as well as donor-to-donor variation. As a solution to these potential drawbacks, presented here is a protocol introducing cryopreservation of freshly isolated PBMs28 for the cell culture model assembly. The aim of this study is to demonstrate the 3D human alveolar epithelial tissue model assembly, including the isolation of PBMs from human buffy coats. The responsiveness to proinflammatory stimuli is compared to the model composed of MDMs and MDDCs differentiated from fresh PBMs or differentiated from frozen/thawed PBMs.
Working with untested human blood samples involves specific care to prevent the potential transmission of infectious diseases, such as HIV (human immunodeficiency virus), hepatitis B, and hepatitis C. Therefore, the use of personal protective measures such as gloves, gowns, masks, and eye protection are crucial and must be in accordance with good laboratory practice principles. These protections reduce the risk of exposing the skin or mucous membranes to potentially infectious fluids. Additionally, for those involved in handling buffy coats and PBMs, vaccination against the hepatitis B virus is mandatory, and blood titer levels of antibodies anti-hepatitis B must be above 100 IU/L (country-specific legislative requirements need to be addressed). In addition, all work must be performed in biosafety level 2 laboratories (country-specific legislative requirements need to be addressed). Standard health and safety precautions associated with working in a laboratory environment and handling mammalian cell culture, including handling of waste, should be adopted when conducting the entire protocol.
The work involving primary monocytes isolated from human blood was approved by the committee of the Federal Office for Public Health Switzerland (reference number: 611-1, Meldung A110635/2) for the Adolphe Merkle Institute.
1. Isolation of peripheral blood monocytes (PBMs) from human buffy coats
NOTE: The following section describes the isolation of immune cells from one 50 mL bag of a buffy coat, purchased from the Swiss Transfusion Centre in Bern, Switzerland.
Preparation of reagents
Prepare 100 mL of magnetic separation buffer per buffy coat: 0.5% [w/v] bovine serum albumin (BSA; in phosphate-buffered saline [PBS]) with 2 mM ethylenediaminetetraacetic acid (EDTA), and adjust to pH = 7.2, sterile filter with 0.22 µm pore size). Keep at 4 °C throughout the procedure.
Prepare the cell culture medium (CCM): RPMI 1640 with 10% [v/v] fetal bovine serum (FBS), 1% [v/v] L-glutamine (here, 2 mM L-glutamine), and 1% [v/v] penicillin-streptomycin (here, 100 units/mL penicillin and 100 µg/mL streptomycin).
NOTE: The required amount of each reagent depends on the number of cells to be seeded in the following steps.
Isolation of PBMs
NOTE: All glass and plasticware needs to be sterilized before use. For safety reasons, the use of plasticware is recommended when handling human blood samples to reduce the risk of injury with glassware.
Use scissors to cut open the hose end of the bag containing the buffy coat.
Distribute the buffy coat by pouring the bag's contents through the bag's duct directly into two conical centrifuge 50 mL tubes (~25 mL each).
Gently pour or pipette PBS into the tubes to reach 50 mL volumes. Mix the contents by turning the tube gently upside down 3x.
Divide the buffy coat-PBS mixture into four new 50 mL conical centrifuge tubes by pipetting 25 mL of the mixture in each fresh tube.
Slowly lay 13 mL of density gradient medium underneath the buffy coat-PBS mixture using a 10 mL serological pipette. Detach the filled pipette from the pipette holder and immediately plug the upper opening of the pipette with a thumb to prevent any additional leaking of the density gradient medium into the buffy coat-PBS mixture.
NOTE: Holding the upper opening with the thumb, place the filled pipette at the bottom of the conical centrifuge tube so that the density gradient medium slowly flows beneath the buffy coat-PBS mixture, leaving approximately 1 mL of the density gradient medium inside the pipette.
Repeat step 1.2.5 with the other three tubes containing the buffy coat-PBS mixture.
Centrifuge all four tubes containing the mixtures for 20 min at 1,000 x g and 25 °C at a slow braking mode. Use holders with protective lids for centrifugation.
Open the lid of each tube, remove the upper layer containing plasma and platelets using a serological pipette, and dispose in a biohazard liquid waste container.
Use a serological pipette for collecting the peripheral blood mononuclear cell layer, which appears as a whitish turbid small fraction (~2–3 mm in thickness) between the plasma and the density gradient medium layers. The pellet contains red blood cells at the bottom. Avoid transferring erythrocytes that form the bottom-most layer. Repeat this for all four tubes.
NOTE: Peripheral blood mononuclear cells consist of PBMs and lymphocytes. PBMs will be separated from lymphocytes later during magnetic CD14+ separation.
Pool peripheral blood mononuclear cells from four tubes into two 50 mL tubes.
Fill up the two tubes with PBS to 50 mL and cover with a lid.
Discard the leftover erythrocytes and plasma from the original four tubes in a biohazard liquid waste container.
Centrifuge the two tubes for 8 min at 500 x g and 18–20 °C at a regular centrifuge speed.
After centrifugation, remove the supernatant with a serological pipette and discard it in a biohazard liquid waste container.
Resuspend the cells with 5 mL of PBS using a serological pipette.
Pool the cell suspensions into one 50 mL conical centrifuge tube and fill to 50 mL with PBS.
Use 5 µL of the cell suspension to count the cells with a cell counter using the trypan blue (45 µL) exclusion method.
Pipette 10 µL of the trypan blue-PBMs solution into a cell counter chamber and count the number of cells per the standard counting protocol. Use Equation 1 to calculate the total cell number, CT.
After counting the cells, centrifuge the 50 mL tube as done in step 1.2.13.
CD14 positive selection
Gently open the lid of each tube, then remove and discard the supernatant using a serological pipette without disturbing the pellet.
Add the calculated amount (Equation 2) of magnetic separation buffer (here, 80 µL of buffer per 1 x 107 total cells) and resuspend the cell pellet by pipetting the solution up and down.
Calculate using Equation 3 (here, 10 µL per 1 x 107 total cells) the corresponding volume of CD14+ magnetic beads and pipette the appropriate volume.
Mix well by pipetting up and down, close the lid, and incubate the solution at 4 °C for 15 min.
Upon incubation, fill the tube up to 50 mL with magnetic separation buffer.
Centrifuge as done in step 1.2.13.
Aspirate and discard the supernatant using a serological pipette without disturbing the cell pellet.
Pipette the corresponding amount of magnetic separation buffer (Equation 4; here, 500 µL of buffer per 1 x 108 cells) and gently mix by pipetting up and down 3x.
Disinfect the magnetic separation station by spraying and wiping it with a sterilizing agent. Place in the laminar flow hood along with the column for magnetic separation.
Place the magnetic separation column in the magnetic field and place an empty 50 mL conical centrifuge tube directly under the column for collecting the wash and unlabeled cells (i.e., waste).
Rinse the magnetic separation column by pipetting 3 mL of magnetic separation buffer into the column. Do not let the column dry out throughout the procedure.
Prepare a 15 mL conical centrifuge tube and pipette 1 mL of magnetic separation buffer.
Apply the cell suspension (prepared in step 1.3.8) to the magnetic separation column. In the 50 mL conical centrifuge tube under the filter, collect unlabeled cells that passed through.
NOTE: Do not exceed 2 x 109 cells per column to avoid blocking of the column.
As soon as the column reservoir is empty (i.e., when the cells have passed through the column), apply 3 mL of magnetic separation buffer using a serological pipette and let it pass through the column. Repeat this 3x.
Remove the magnetic separation column from the magnetic separator by gently pulling with hands, then place it into a 15 mL tube containing pre-pipetted 1 mL of magnetic separation buffer (prepared in step 1.3.12).
Add 5 mL of magnetic separation buffer to the column and flush out the magnetically labeled cells by firmly pushing the plunger into the column.
Reagent preparation for MDM and MDDC differentiation
Count the cells with a cell counter using the trypan blue exclusion method as done in step 1.2.17.
Calculate the required volumes of CCM or FBS for further steps as follows: either the volume of CCM corresponding to a cell density of 1 x 106 cells/mL (step 1.5.1), or the volume of FBS corresponding to a cell density of 6 x 106 cells per 0.9 mL of FBS (step 1.6.3).
Close the lid, place the tube in the centrifuge, and centrifuge as done in step 1.2.13. Remove and discard the supernatant without disturbing the cell pellet. Proceed to step 1.5 for cell seeding or step 1.6 for cell freezing.
PBM seeding and differentiation into MDMs and MDDCs
Resuspend the cell pellet in the calculated volume of CCM as calculated in step 1.4.2 (here, a final concentration of 1 x 106 cells/mL) by pipetting up and down 3x.
Pipette the number of cells intended to differentiate into MDMs and MDDCs in separate conical centrifuge tubes using a serological pipette.
Pipette differentiating factors to the CCM with PBMs and mix well by pipetting up and down. Differentiating factors are applied as follows:
For MDDCs: final concentration of 10 ng/mL interleukin-4 (IL-4) and 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF).
For MDMs: final concentration of 10 ng/mL macrophage colony-stimulating factor (M-CSF).
Pipette the cell suspensions in CCM with the added differentiating factors into 6 well plates by distributing 3 mL of the suspension per well (corresponds to 3 x 106 cells/well, i.e., 1 x 106 cells/mL).
Place the 6 well plates in a cell culture incubator (37 °C, 5% CO2) and let them differentiate for 6 days without refreshing the CCM.
NOTE: Differentiation ranges from 5–8 days depending on the local availability of buffy coats and experimental setup, provided that the differentiation efficiency is determined using the appropriate techniques (refer to the discussion section).
PBM freezing
Resuspend the cell pellet in a cryoprotective medium (here, FBS and dimethyl sulfoxide [DMSO; cytotoxic]) at a ratio of 9:1 (v/v) by pipetting a volume of prewarmed FBS. This corresponds to a final cell concentration of 6 x 106 cells/mL, considering a further addition of 10% DMSO (v/v).
Mark the desired number of cryovials in the laminar flow hood (i.e., record the date, isolation code, and number of cells).
Pipette 0.9 mL of cell suspension in pure FBS (here, 6 x 106 cells in 0.9 mL of FBS) to each cryovial. Subsequently, slowly pipette 0.1 mL of DMSO and mix the suspension well by turning the cryovials up and down 3x.
Transfer the cryovials to a cell-freezing container and immediately set it to -80 °C for 24 h.
After 24 h, remove the cryovials from the -80 °C freezer and container and place them into the liquid nitrogen tank suitable for cell storage.
PBM thawing and differentiation into MDMs and MDDCs
Warm all the required reagents to 37 °C in a water bath (~20–30 min).
Prepare the appropriate number of 6 well plates corresponding to the number of thawed cells (here, one plate per 1.8 x 107 cells, i.e. 3 cryovials). Pipette 2 mL of CCM to each well under aseptic conditions. Place plates in the incubator (5 % CO2, 37 °C) for 15 min to allow the pH to equilibrate.
Take the required amount of cryovials with frozen cells from a liquid nitrogen tank and gently swirl them in a 37 °C water bath (1–2 min) to ensure uniform thawing of the cell suspension.
Remove the cryovial from the water bath and decontaminate with a sterilizing agent, ensuring that the agent does not interact with the lid and O-ring.
NOTE: Hereon, all steps must be completed under aseptic conditions.
Prepare the appropriate number of 15 mL conical centrifuge tubes corresponding to the number of cryovials to be thawed (here, 6 x 106 cells/tube). Pipette 9 mL of prewarmed CCM into each tube.
Pipette slowly (drop-by-drop) the contents of the cryovial into a tube containing CCM. Close the lid, repeat for each tube, and centrifuge at 200 x g for 5 min at a regular centrifuge speed.
Discard the supernatant without disturbing the pellet.
Resuspend the pellet from each tube (which contains cells from one cryovial) in 2 mL of prewarmed CCM by pipetting up and down using a serological pipette (cell density corresponding to 3 x 106 cells/mL).
From each tube, pipette the resuspended cells into two wells (1 mL per well) of a 6 well plate containing 2 mL of previously prepared CCM to reach a cell density of 3 x 106 cells/well (corresponding to a final concentration of 1 x 106 cells/mL). Repeat this for all other tubes.
Proceed with differentiation as described in section 1.5.3.
2. Triple cell coculture model of human alveolar epithelial tissue
NOTE: This section provides instructions on volumes and cell numbers corresponding to 12 well plate inserts. Figure 1 summarizes a proposed timeline for model assembly.
Epithelial cell (A549 cell line) seeding
Culture the epithelial cells according to the recommendations provided by the supplier (ATTC). Briefly, subculture the cells in CCM at 80% cell confluency (approximately, 2x–3x per week).
NOTE: Subculture A549 for at least four passages before the coculture model composition, using A549 cells in a passage range of 5–25.
Pipette 1.5 mL of prewarmed CCM into 12 well plates (the number of wells corresponds to the desired number of models).
Place individual 12 well cell culture inserts into wells of a 12 well plate using sterilized tweezers.
Detach the cells from a flask according to the subcultivation protocol (i.e., using a detaching agent, remove the agent by centrifugation as done in step 1.2.13). Resuspend in the appropriate volume of CCM corresponding to the final cell concentration of A549 (here, 50 x 104 cells/mL; 0.5 mL of cell suspension per insert, i.e. 25 x 104 cells/insert, which corresponds to seeding density of 27.8 x 104 cells/cm2).
Pipette 0.5 mL of the cell suspension (i.e., 25 x 104 cells/insert) into the apical side of the insert using a 1 mL pipette.
Cover the plates with lids and place them in a cell culture incubator (37 °C, 5 % CO2) for 4 days.
NOTE: Regularly check the confluency of A549 cells under a phase-contrast microscope.
MDDC seeding
Aspirate CCM with unattached cells in the 6 well plates containing MDDCs.
Add 1 mL of fresh prewarmed CCM to each well.
Use a cell scraper, detach (scrape) adherent MDDCs from each well, gently wash the wells with the existing 1 mL of CCM 3x, and combine them into one conical centrifuge tube.
Count the cells with a cell counter using the trypan blue exclusion method using 10 μL of cell suspension and 10 μL of trypan blue solution.
Centrifuge the cell suspension as done in step 1.2.13.
Calculate the CCM volume required for resuspension (Equation 5):
Required MDDC density = 42 x 104 cells/mL; each insert requires 6.3 x 104 cells, which corresponds to a seeded cell density of 7 x 104 cell/cm2 (here, 150 µL added onto 0.9 cm2 in the step 2.2.10.).
CCM volume for cell resuspension (Vm; Equation 5):
Gently aspirate and discard the CCM from the upper chamber of 12 well plates with growing A549 on the inserts.
Place the inserts with A549 cells in an upside-down position in a sterile Petri dish using sterilized tweezers. Prepare a conical centrifuge tube (50 mL) with PBS and pre-moisten a cell scraper.
Scrape off A549 cells from the basal surface of the insert (i.e., the upper part at the upside-down position), which should grow through the pores of the inserts.
NOTE: Rinse the scraper with PBS (prepared in a tube) between scraping individual samples and keep it wet throughout the procedure.
Upon centrifugation (step 2.2.5), aspirate and discard the supernatant, then redisperse the MDDC pellet in the calculated amount of CCM (step 2.2.6) and pipette up and down 3x.
Pipette 150 µL of the cell suspension on top of each insert so that the entire basal surface of the insert is equally covered with the liquid and not containing bubbles.
Cover the dish with a lid and place in a cell culture incubator for 70 min. Aspirate the CCM from the cell culture plates (where the inserts have initially been placed), discard it in a biohazard liquid waste, and pipette 1.5 mL of fresh CCM into each well. Cover the plate with a lid and place in the cell incubator (37 °C, 5% CO2).
NOTE: Do not exceed the time period mentioned above to avoid drying out the cells.
After the incubation, carefully hold each insert with sterilized tweezers and place them to the plates containing CCM in an ordinary position. Cover the plate with a lid and return it to the cell incubator (37°C, 5% CO2).
Macrophage (MDM) seeding
Take 6 well plates containing predifferentiated MDMs. Place them from the cell incubator to a laminar flow hood.
Aspirate and discard CCM with unattached MDMs grown in 6 well plates, and pipette 1 mL of fresh prewarmed CCM in each well.
Using a cell scraper, gently remove adherent MDMs from individual wells (as done in step 2.2.3 for the MDDCs).
Pipette 10 µL of trypan blue into a well or tube and add 10 µL of the MDM suspension to achieve the final dilution of 1:1 (v/v). Count the number of MDMs using the appropriate counting protocol.
Calculate the required volume (Equation 6):
Required MDM density = 2.5 x 104 cells/mL in CCM (here, each insert requires 1.25 x 104 cells in 0.5 mL CCM, which corresponds to a seeded cell density of 1.4 x 104 cell/cm2).
Upon centrifugation, aspirate and discard the supernatant, redisperse the MDM pellet in the calculated amount of CCM (step 2.3.6), and pipette up and down 3x.
Carefully pipette 0.5 mL of the MDM suspension (prepared in step 2.3.7) on the wall of the cell culture inserts with A549 and MDDCs (not directly on epithelial cells) using a 1 mL pipette. Cover plates with lids and place in a cell culture incubator (37 °C, 5 % CO2) for 24 h.
Transfer of coculture model to air-liquid interface (ALI)
Upon the end of the 24 h (±2 h) incubation period of the assembled model in a cell culture incubator, aspirate and discard CCM from both apical and basal parts of cell culture inserts and from the wells.
Using sterilized tweezers, lift individual inserts from the wells and pipette 0.6 mL of fresh prewarmed CCM to each well using a 1 mL pipette. Do not add CCM to the apical side of the insert.
Cover plates with lids and place in a cell culture incubator (37 °C, 5 % CO2) for 24 h prior further use.
3. Exposure to selected positive controls (known stimuli for inducing proinflammatory response)
NOTE: Exposure of the coculture models to a known proinflammatory stimulus endotoxin lipopolysaccharide (LPS)7 and proinflammatory cytokine tumor necrosis factor α (TNF-α)7 is used to illustrate responsiveness of the model. Furthermore, exposure to a detergent (Triton X-100) is used to confirm the sensitivity of a lactate dehydrogenase (LDH) assay.
Prepare the positive control solutions: LPS stock (1 mg/mL in distilled water), TNF-α stock (100 μg/mL in distilled water) and Triton-X 100 (2% [v/v] in PBS).
Upon 24 h incubation of the coculture model at ALI conditions, aspirate and discard the supernatant from the basal compartment. Using sterilized tweezers, lift individual inserts from the wells and pipette 0.6 mL of fresh prewarmed CCM into each well.
Prepare individual positive controls working solutions by diluting the stocks in CCM in conical centrifuge tubes as follows: 1 μg/mL LPS, 1 μg/mL TNF-α, and 0.2% Triton-X 100. The volumes correspond to the number of tested inserts (here, 100 μL/insert). Mix the solutions well by pipetting up and down 3x.
Apply 100 μL of each positive control solution by slowly pipetting onto the wall of the cell culture insert. Cover the well plate with a lid and place in the cell culture incubator (37 °C, 5% CO2) for 24 h. Upon incubation, aspirate and discard the liquid at the apical side of the insert by holding individual inserts using tweezers.
Collect CCM in the basal compartments and store at 1) 4 °C for further LDH analysis, denoting cell membrane rupture-mediated cytotoxicity, and/or 2) store at -80 °C for further analysis of protein release via enzyme-linked immunosorbent assay (ELISA). Run the assays according to the kit supplier recommendations.
Upon the removal of CCM, wash the inserts with PBS 3x and fix the cells on cell culture inserts in 4% [w/v] paraformaldehyde (in PBS, 15 min at room temperature) by ensuring both insert sides are well-covered with PFA solution. Subsequently wash 3x with PBS to remove PFA. Store the samples submerged in PBS at 4 °C for further immunostaining (an example of this method has been described previously17).
Representative Results
Human lung coculture models, composed of alveolar epithelial cells and immune cells, were assembled either from fresh or frozen MDDCs and MDMs progenitors (here, human peripheral blood-derived monocytes). As presented in Figure 1, A549 cells were seeded 3 days after the first section involving monocyte isolation/thawing. After 6 days of differentiation, the differentiated MDMs appeared round-shaped, whereas MDDCs formed a more elongated shape with observable protrusions. They also appeared as agglomerates, especially when differentiated from fresh monocytes (Figure 2, Figure 3). Epithelial cells formed a dense cell layer of cells after 3 days of growth on membrane inserts (Figure 4), when the cocultures were assembled. After 24 h of assembling and an additional 24 h of being subjected to ALI conditions, the cocultures were prepared for exposures.
Responsiveness of the 3D cell culture models was investigated upon exposure to known proinflammatory stimuli using a pseudo-ALI approach, as described previously29. The proinflammatory stimuli, LPS and TNF-α, were added in low volumes (100 μL) onto the apical surface of the air-exposed cell model. In parallel, the absence of membrane rupture as a measure of cytotoxicity was assessed via LDH assay. A significant increase in LDH release in CCM of the basal compartment was observed upon exposure to the positive control for membrane rupture, a detergent Triton-X 100 (Figure 5). These results proved responsiveness of the model to a cytotoxic substance, whereas no increase in LDH release was observed upon apical stimulation with TNF-α or LPS.
A possible reason for the different measured values of LDH in the samples assembled with either fresh or previously frozen PBMs can be attributed to the sample storage. Samples from fresh PBMs were stored for a longer time at -80 °C; therefore, the activity of LDH enzyme may drop. Notably, LDH is stable for only up to 4 days in CCM; thus, it is recommended to perform the assay the latest 2 days after collecting the supernatants. Alternatively, it is possible to freeze down the supernatants directly after collection. However, it is important to consider that freezing can decrease the enzymatic activity of LDH.
Secretion of proinflammatory mediators (here, TNF-α and interleukins 6 [IL-6] and 8 [IL-8]) into the basal CCM was quantified via ELISA. Statistically significant (p < 0.05, one-way ANOVA) increases in the release of IL-6 and IL-8 were observed in both LPS- and TNF-α- treated samples compared to respective untreated cells, as well as in the cell culture models assembled from either PBMs source (Figure 6). Although concentrations (pg/mL) of all the tested cytokines in the basal CCM were higher in the cocultures composed of fresh PBMs, differences among the two cocultures and monocultures were not statistically significant (p > 0.05) (Figure 6). To confirm the added value of coculture models with respect to a 2D epithelial cell culture, A549 monocultures were also exposed to LPS or TNF-α. As expected, the release of all investigated mediators from A549 monocultures was lower compared to both coculture models; although, the difference between them was not statistically significant (p > 0.05, one-way ANOVA).
Cellular morphology of the 3D human alveolar epithelial tissue barrier was assessed via confocal laser scanning microscopy (LSM). To visualize the composition of each model, macrophages within the coculture models (MDMs) were stained with mature macrophage marker 25F9. MDDCs were stained with CD83, which is an important marker for activated dendritic cells30. Regarding cellular morphology, no difference was observed between the coculture models using MDMs and MDDCs from fresh PBMs compared to those using thawed PBMs. In LPS- and TNF-α-exposed cocultures, both composed of fresh and frozen immune cells, a disrupted epithelial layer in LSM images was observed, which was not the case in untreated cells (Figure 7, Figure 8).
Figure 1: Schematic timeline of the protocol. Presentation of the 3D coculture model preparation, assembly, and application (exposure to a tested substance). ALI = air-liquid interface, MDDCs = monocyte-derived dendritic cells, MDMs = monocyte-derived macrophages, PBMs = peripheral blood monocytes. Please click here to view a larger version of this figure.
Figure 2: MDMs and MDDCs differentiated from fresh PBMs. Phase-contrast microscopy image of differentiated (A) MDMs and (B) MDDCs from fresh PBMs (6 days after cell isolation). MDMs are round-shaped, while MDDCs are often observed as agglomerates. Please click here to view a larger version of this figure.
Figure 3: MDMs and MDDCs differentiated from frozen PBMs. Phase-contrast microscopy image of differentiated (A) MDMs and (B) MDDCs from thawed PBMs (6 days after thawing). MDMs are round-shaped, but some elongated cells can be observed. MDDCs also appear round-shaped with protrusions. Please click here to view a larger version of this figure.
Figure 4: Epithelial cells growth on membrane inserts. Phase-contrast microscopy image of confluent A549 growing on a membrane insert 4 days after seeding, forming a dense layer of cells. Please click here to view a larger version of this figure.
Figure 5: Cytotoxicity results investigated via membrane rupture-based (LDH) assay. The data is presented as a fold increase over untreated cells (mean ± SD, n = 3, asterisk denotes statistically significant increase compared to untreated cells, **p < 0.01, ****p < 0.0001). In green models MDMs and MDDCs from fresh PBMs are represented, and in purple models assembled from thawed PBMs are represented. Please click here to view a larger version of this figure.
Figure 6: Proinflammatory reactions in the cocultures and monocultures. The proinflammatory mediators (TNF-α, IL-6, and IL-8) release in the cocultures upon 24 h challenge with LPS or TNF-α. The data is presented as relative to untreated cells (mean ± SD, n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001). In green models MDMs and MDDCs from fresh PBMs are represented, and in purple models assembled from thawed PBMs are represented. Grey represents A549 monocultures. Please click here to view a larger version of this figure.
Figure 7: Morphology of cocultures composed of fresh immune cells. LSM images of apical and basal sides of the coculture model with xz projections of apical sides of the model using MDMs and MDDCs from fresh PBMs. Cyan represents nuclei (DAPI), magenta represents cytoskeleton (rhodamine-phalloidin), white represents MDMs (25F9), and green represents MDDCs (CD 83). The white arrow denotes MDM, whereas the green arrow denotes MDDC. Please click here to view a larger version of this figure.
Figure 8: Morphology of cocultures composed of frozen immune cells. LSM images of the apical side of the coculture model with corresponding xz projections, and basal side of the model using MDMs and MDDCs from thawed PBMs. Cyan represents nuclei (DAPI), magenta represents cytoskeleton (rhodamine-phalloidin), white represents MDMs (25F9), and green represents MDDCs (CD 83). The white arrow denotes MDM, whereas the green arrow denotes MDDC. Please click here to view a larger version of this figure.
Emerging production of novel materials, including chemicals and drugs, gradually increases the need for predictive in vitro models. To comply with the three principles of replacement, reduction, and refinement of animal testing32, in vitro cell models have become powerful tools regarding the replacement and reduction aspect for elucidating mechanisms of a drug's or material's action8,9,10,11. Presented here is a detailed protocol of assembling the multicellular model using immune cells that are either freshly isolated or thawed from previously frozen monocytes. Also described is the cultivation of the model at ALI. Finally, the protocol illustrates an example of exposure to proinflammatory stimuli and compares the response of the two models containing either fresh or frozen monocytes.
Various studies have been performed to confirm and justify the added value of the enhanced complexity of the models grown and exposed under ALI conditions compared to conventional submerged exposure7,22,31. Observation of the higher proinflammatory response in cocultures compared to monocultures of epithelial cells confirm a previous study. The study used the presented coculture model (stimulated with LPS) and showed a higher response at gene expression levels of TNF and IL1B compared to the A549 monoculture equivalent model7. On the other hand, both models showed higher variations within measured proinflammatory mediator release values compared to A549 monocultures. This can be explained by the use of immune cells from different donors (buffy coats) within biological repetitions (i.e., one repetition, one donor), as previously shown7. If desired, the variations among the replicates can be overcome by 1) using thawed PBMs from the same donor or 2) pooling PBMs from different donors prior to freezing the cells, then subsequent usage of the same pool in each repetition. Including more biological repetitions is also recommended.
The cell-freezing technique can be considered as a critical step; however, it is a common laboratory procedure for preserving cells for phenotypic and functional analysis. Various studies have demonstrated that the quality of frozen PBMs is vital to their survival, and an appropriate freezing technique is key to the success of subsequent assays with the same cells28,32. Modification of the protocol can be performed by freezing PBMs, which provides flexibility in the experimental setup, as the availability of buffy coats is usually limited. Another advantage of using frozen PBMs (in several vials) over freshly isolated ones is that they can be used in subsequent experiments even after 1 year. This decreases the potential issue of donor-to-donor variability if this is a desired or required parameter in an experimental.
Outcomes of an interlaboratory comparison performed after up to 13 months show that PBMs, when properly stored in a liquid nitrogen tank, can be used over a long period without any effect on cell viability or cell recovery33. Longer storage times (over 1 year) may be possible upon careful validation of cell viability and cell responsiveness before performing an experiment. Also, the temperature in the liquid nitrogen tank must remain stable at all times. The main factor affecting the viability of cryopreserved PBMs was found to be DMSO concentration, with an optimal concentration of 10%–20 % (v/v)28. To minimize potentially harmful effects of freezing, different sources of proteins, FBS or BSA (with a broad range of concentration from 40% up to 100 %34) are often added to the freezing medium as natural protective components that can increase cell survival.
Due to the high cytotoxic potential of DMSO, it is recommended first to disperse PBMs in FBS, then add DMSO to PBMs already dispersed in FBS. Notably, although higher FBS concentrations (>40%) did not show any improvement in cell viability, at the same time, they did not cause harm to the cells28. Nevertheless, freezing monocytes is a possible approach to overcoming issues of limited buffy coat availability. However, if the use of MDDCs and MDMs from fresh PBMs is desired, the immune cells can be differentiated and used 5–8 days after isolation7,16,17,35,36,37. If experimental planning allows, at least 6 days of differentiation in both MDDCs and MDMs is recommended. However, consistency among different repetitions in the same experiment, along with routine inspections of their specific surface marker expressions, are crucial. The responsiveness to a proinflammatory stimulus, such as LPS, after the differentiation time should also be regularly checked.
Many investigations using the A549 cell line have been performed at ALI, either as a monoculture or combined with other cell types (macrophages, dendritic cells, or fibroblasts) into 3D coculture model22,24,29,38. Using this 3D coculture model, the cytotoxicity, oxidative stress, or proinflammatory effects of (nano-)materials have been investigated for up to 72 h1,17,21,24,29. The model's resemblance to in vivo tissue has previously been investigated based on confocal laser scanning imaging of the model16. When assembling the model, it is important to consider both cell proliferation (which can affect A549 in the model presented here) as well as performance of the primary (not proliferating) immune cells (here, MDDCs and MDMs). It is also important to consider that not all the CD14 positive monocytes differentiate into MDDCs and MDMs, and that the cells can be present in both attached and suspended forms. Based on the nature of the coculture assembly (here, both cell types need to attach to the existing epithelial layer), it is recommended to use only the adherent sub-populations of both immune cell types. Additionally, routine analyses of monocytes, MDDC and MDM monoculture responsiveness to LPS, and expression of specific surface markers (CD14, CD163, CD86, CD93, or CD206, data not shown) have suggested that 6 and 7 days of differentiation are the optimum timepoints.
Although a realistic number of alveolar epithelial cells in human lungs corresponds to ~160,000 cells/cm2, the number of A549 cells counted in the model is ~1,000,000 cells/cm2 after 9 days cultured on the insert16,18. Thus, this in vitro model's limitations need to be considered. First, the density of epithelial cells was established based on their ability to form a confluent layer on the growing membrane. It is also important to mention that the A549 represents an epithelial type II cell with a cuboidal form, contrary to epithelial type I cells, which are flat and outspread. On the other hand, the required number of immune cells was established based on the literature and presented in this protocol as cell number/surface area39,40,41. The cell density of MDDCs in the range of 400 cells/mm2 (4 cells/cm2)16 is comparable to the steady-state cell density of 500–750 cells/mm2 (5- 7 cells/cm2) reported from in vivo studies39. The density of MDMs in this model is within the same range of in vivo situation in the human alveolar region40.
Mature macrophage marker staining (25F9) was observed both in the apical side (where MDMs are present) as well as basal side (i.e., at the site of dendritic cells). Translocation of immune cells through the membrane inserts pores is possible and has also been observed using this model16, which may explain the observed differences in staining intensities. However, another possible explanation is that the mature macrophage marker can also be expressed on dendritic cells, but the expression is highly donor-specific42. Also, the intensity of the 25F9 expression is much higher in MDMs (Figure 7, Figure 8). Both proinflammatory stimuli (LPS and TNF-α) affected the integrity of the pulmonary epithelial barrier in both cocultures (Figure 7, Figure 8). This was expected based on previous publications43,44 showing that proinflammatory cytokines and bacterial products disrupt the integrity of epithelial barriers.
The 3D multicellular model of the human alveolar epithelium, established and characterized previously17, has served as a powerful and useful tool for assessing biological responses (i.e., acute proinflammatory reactions, oxidative stress response, particle distribution, and cellular communication) in vitro21,24,25,45. The results confirm the responsivness of coculture models to proinflammatory stimuli (here, LPS and TNF-α). The response was slightly increased when using immune cells from fresh PBMs; however, there was no statistically significant difference between cocultures using fresh vs. thawed PBMs. Furthermore, the proinflammatory reactions of both coculture models were higher than those of epithelial cell monocultures cultivated under the same (ALI) conditions. In summary, the protocol describes the assembly of a 3D human alveolar epithelial tissue coculture model using either fresh or thawed PBMs for differentiation into MDMs and MDDCs. It is shown that both models are highly responsive to proinflammatory stimuli; therefore, they can serve as powerful tools for potential hazard and toxicity assessments.
The authors have nothing to disclose.
The authors would like to thank Dr. Miguel Spuch-Calvar for the coculture scheme in Figure 3 and to Dr. Bedia Begum Karakocak for critical reading. This study was supported by the PATROLS project, European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 760813, and by the Adolphe Merkle Foundation. B.D. thanks the Peter und Traudl Engelhorn foundation for financial support.
Name Company Catalog Number Comments
Benchmark microplate reader does not have to be specific, for example BioRad, Cressier, Switzerland
Cell culture Incubator does not have to be specific
Cell freezing container (for example Mr. Frosty) does not have to be specific
Centrifuge does not have to be specific
Confocal laser scanning microscope does not have to be specific, for example Zeiss LSM 710 meta
Heamatocytometer, or automatic cell counter does not have to be specific
Laminar bio-safety hood class II does not have to be specific
MultiStand Macs (Macs Cell Separator) Miltenyi, Germany 130-042-303
pH meter does not have to be specific
Phase contrast inverted light microscope does not have to be specific
Pipette boy, pipettors (different volumes) do not have to be specific
Scissors do not have to be specific
Vacuum pump does not have to be specific
Water bath does not have to be specific
Disposable small equipment/glassware Catalogue Number
15 mL and 50 mL conical centrifuge tubes does not have to be specific
6- and 12-well cell culture plates, flat bottom, low evaporation lid, sterile Falcon, Switzerland 353046 and 353043
Cell culture inserts, transparent PET membrane, 12-well, 3 μm pore size Falcon, Switzerland 353181
Cell scrapper does not have to be specific, for example VWR, Switzerland 353085
Cryovials do not have to be specific
Glass autoclaved Petri Dishes do not have to be specific
LS Columns Miltenyi, Germany 130-042-401
Sterile filtration cup for vacuum filtration, 0.2 μm pore size does not have to be specific, for example VWR, Switzerland 10040-446
Sterile Lab Bottle compatible with Filtration cup (min. 100 mL) does not have to be specific
Sterile pipettes do not have to be specific
Bovine serum albumine (BSA) Sigma-Aldrich, Switzerland A7030-100g
CD14+ MicroBeads human - magnetic beads Miltenyi, Germany 130-097-052
Deattachnig agent Trypsin-EDTA, 0.05%, phenol red Gibco, Switzerland 25300054
Density gradient medium Lymphoprep Alere Technologies AS, Norway 1114547
Dimethyl Sulfoxide (DMSO) Sigma Aldrich, Switzerland D2438
Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich, Switzerland E6758-100g
Fetal bovine serum (heat inactivated) Gibco, Switzerland 10270-106
Human granulocyte-macrophage colony-stimulating factor (GM-CSF), premium grade Miltenyi, Germany 130-093-864
Human Interleukin 4 (IL-4), premium grade Miltenyi, Germany 130-095-373
Human macrophage colony-stimulating factor (M-CSF), premium grade Miltenyi, Germany 130-096-485
L-glutamine Gibco, Switzerland 25030-024
Lipopolysaccharid (LPS) from Escherichia coli Sigma-Aldrich, Switzerland 4524-5mg
Paraformaldehyde (PFA) Sigma-Aldrich, Switzerland 158127
Penicilin-Streptomycin Gibco, Switzerland 15140-122
Phosphate Buffer Saline (PBS) Gibco, Switzerland 10010-015
Roswell Park Memorial Institute-1640 Medium (RPMI) Gibco, Switzerland 42401-018
Triton X-100 Sigma-Aldrich, Switzerland T8787
Trypan blue solution (0.4%) Sigma Aldrich, Switzerland
Tumor necrosis factor alpha (TNF-α) Immunotools 11343015
Assays used for cytotoxicity, (pro-)inflammatory response
Cytotoxicity Detection Kit (LDH) Roche, Switzerland 11644793001
Human IL-6 DuoSet ELISA R&D, Biotechne, Switzerland DY206
Human IL-8/CXCL8 DuoSet ELISA R&D, Biotechne, Switzerland DY208
4′,6-diamidino-2-phenylindole (DAPI), concentration 2 μg/mL Sigma-Aldrich, Switzerland 10236276001
Goat anti-mouse IgG (H+L) Alexa Fluor 647 conjugated, concentration 20 μg/mL Abcam, UK ab150115
Goat anti-rabbit IgG antibody (H+L) Dylight 488 conjugated, concentration 10 μg/mL Agrisera, Sweden AS09 633
Mature Macrophage Marker Monoclonal Antibody, concentration 50 μg/mL eBioScience, Thermo Fischer, Switzerland 14-0115-82
Phalloidin rhodamine, concentration 0.264 µM Molecular Probes, Life Technologies, Switzerland R415
Recombinant Anti-CD83 antibody, 1:50 dillution Abcam, UK ab244204
DOWNLOAD MATERIALS LIST
Rothen-Rutishauser, B., Blank, F., Mühlfeld, C., Gehr, P. In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter. Expert Opinion on Drug Metabolism and Toxicology. 4, (8), 1075-1089 (2008).
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Ochs, M., Weibel, E. R., et al. Ch. 2: Functional Design of the Human Lung for Gas Exchange . Fishman's Pulmonary Diseases and Disorders, 5e. Grippi, M. A., et al. McGraw-Hill Education. (2008).
Foster, K. A., Oster, C. G., Mayer, M. M., Avery, M. L., Audus, K. L. Characterization of the A549 Cell Line as a Type II Pulmonary Epithelial Cell Model for Drug Metabolism. Experimental Cell Research. 243, (2), 359-366 (1998).
Guo, X. Y., Lu, M., Chen, X. Q., He, F. D., Li, A. Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent. Asian Pacific Journal of Tropical Medicine. 9, (6), 582-586 (2016).
Cooper, J. R., et al. Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype. PLoS ONE. 11, (10), 0164438 (2016).
Bisig, C., Voss, C., Petri-Fink, A., Rothen-Rutishauser, B. The crux of positive controls - Proinflammatory responses in lung cell models. Toxicology In Vitro. 54, 189-193 (2019).
Rothen-Rutishauser, B., et al. A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles. ALTEX. 25, (2008).
Braakhuis, H. M., et al. Progress and future of in vitro models to study translocation of nanoparticles. Archives of Toxicology. 89, (9), 1469-1495 (2015).
Thai, P., Chen, Y., Dolganov, G., Wu, R. Differential regulation of MUC5AC/Muc5ac and hCLCA-1/mGob-5 expression in airway epithelium. American Journal of Respiratory Cell and Molecular Biology. 33, (6), 523-530 (2005).
Wu, J., et al. Characterization of air-liquid interface culture of A549 alveolar epithelial cells. Brazilian Journal of Medical and Biological Research. 51, (2), 6950 (2017).
Shapiro, D. I., Nardone, L. L., Rooney, S. A., Motoyama, E. K., Munoz, J. L. Phospholipid biosynthesis and secretion by a cell line (A549) which resembles type II aleveolar epithelial cells. Biochimica and Biophysica Acta. 530, (2), 197-207 (1978).
Balis, J., Bumgarner, S. D., Paciga, J. E., Paterson, J. F., Shelley, S. A. Synthesis of lung surfactant-associated glycoproteins by A549 cells: description of an in vitro model for human type II cell dysfunction. Experimental Lung Research. 6, (3-4), 197-213 (1984).
Schurch, S., Gehr, P., Im Hof, V., Geiser, M., Green, F. Surfactant displaces particles toward the epithelium in airways and alveoli. Respiration Physiology. 80, (1), 17-32 (1990).
Gehr, P., Schurch, S., Berthiaume, Y., Hof, V. I., Geiser, M. Particle Retention in Airways by Surfactant. Journal of Aerosol Medicine. 3, (1), 27-43 (2009).
Blank, F., Rothen-Rutishauser, B., Gehr, P. Dendritic Cells and Macrophages Form a Transepithelial Network against Foreign Particulate Antigens. American Journal of Respiratory Cell and Molecular Biology. 36, (6), (2007).
Rothen-Rutishauser, B. M., Kiama, S. G., Gehr, P. A three-dimensional cellular model of the human respiratory tract to study the interaction with particles. American Journal of Respiratory Cell and Molecular Biology. 32, (2005).
Blank, F., Rothen-Rutishauser, B. M., Schurch, S., Gehr, P. An optimized in vitro model of the respiratory tract wall to study particle cell interactions. Journal of Aerosol Medicine. 19, (2006).
Jardine, L., et al. Lipopolysaccharide inhalation recruits monocytes and dendritic cell subsets to the alveolar airspace. Nature Communications. 10, (1), 1999 (2019).
Kopf, M., Schneider, C., Nobs, S. P. The development and function of lung-resident macrophages and dendritic cells. Nature Immunology. 16, (1), 36-44 (2015).
Chortarea, S., et al. Repeated exposure to carbon nanotube-based aerosols does not affect the functional properties of a 3D human epithelial airway model. Nanotoxicology. 9, (8), 983-993 (2015).
Hilton, G., Barosova, H., Petri-Fink, A., Rothen-Rutishauser, B., Bereman, M. Leveraging proteomics to compare submerged versus air-liquid interface carbon nanotube exposure to a 3D lung cell model. Toxicology In Vitro. 54, 58-66 (2019).
Brandenberger, C., et al. Effects and uptake of gold nanoparticles deposited at the air-liquid interface of a human epithelial airway model. Toxicology and Applied Pharmacology. 242, (2010).
Drasler, B., et al. Single exposure to aerosolized graphene oxide and graphene nanoplatelets did not initiate an acute biological response in a 3D human lung model. Carbon. 137, 125-135 (2018).
Durantie, E., et al. Carbon nanodots: Opportunities and limitations to study their biodistribution at the human lung epithelial tissue barrier. Biointerphases. 13, (2018).
Brandenberger, C., et al. Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles. Small. 6, (15), 1669-1678 (2010).
Tomašek, I., et al. Combined exposure of diesel exhaust particles and respirable Soufrière Hills volcanic ash causes a (pro-)inflammatory response in an in vitro multicellular epithelial tissue barrier model. Particle and Fibre Toxicology. 13, (1), 67 (2016).
Nazarpour, R., et al. Optimization of Human Peripheral Blood Mononuclear Cells (PBMCs) Cryopreservation. International Journal of Molecular and Cellular Medicine. 1, (2), 88-93 (2012).
Endes, C., et al. An in vitro testing strategy towards mimicking the inhalation of high aspect ratio nanoparticles. Particle and Fibre Toxicology. 11, (1), (2014).
Ju, X., et al. The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83+-Activated T Cell Population. Journal of Immunology. 197, (12), 4613-4625 (2016).
Lenz, A. G., et al. Inflammatory and Oxidative Stress Responses of an Alveolar Epithelial Cell Line to Airborne Zinc Oxide Nanoparticles at the Air-Liquid Interface: A Comparison with Conventional, Submerged Cell-Culture Conditions. BioMed Research International. 12 (2013).
Germann, A., Schulz, J. C., Kemp-Kamke, B., Zimmermann, H., von Briesen, H. Standardized serum-free cryomedia maintain peripheral blood mononuclear cell viability, recovery, and antigen-specific T-cell response compared to fetal calf serum-based medium. Biopreservation and Biobanking. 9, (3), 229-236 (2011).
Weinberg, A., et al. Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization. Clinical and Vaccine Immunology. 16, (8), 1176 (2009).
Freshney, R. I. Culture of animal cells: a manual of basic technique. Freshney, R. I. Wiley-Liss Inc. 321-334 (2005).
Lehmann, A. B. C., Blank, F., Gehr, P., Rothen-Rutishauser, B. Alternatives to animal testing. Yarmush, M. L., Langer, R. S. Artech House. 239-260 (2010).
Steiner, S., et al. Reduction in (pro-)inflammatory responses of lung cells exposed in to diesel exhaust treated with a non-catalyzed diesel particle filter. Atmospheric Environment. 81, 117-124 (2013).
Martinez, F. O., Gordon, S., Locati, M., Mantovani, A. Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Gene Expression. The Journal of Immunology. 177, (10), 7303 (2006).
Chortarea, S., et al. Profibrotic activity of multi-walled carbon nanotubes upon prolonged exposures in different human lung cell types. Applied In Vitro Toxicology. 5, (1), (2019).
Holt, P. G. Pulmonary Dendritic Cells in Local Immunity to Inert and Pathogenic Antigens in the Respiratory Tract. Proceedings of the American Thoracic Society. 2, (2), 116-120 (2005).
Pinkerton, K. E., Gehr, P., Castañeda, A., Crapo, J. D. Comparative Biology of the Normal Lung (Second Edition). Parent, R. A. Academic Press. 105-117 (2015).
Crapo, J., Barry, B., Gehr, P., Bachofen, M., Weibel, E. R. Cell number and cell characteristics of the normal human lung. American Review of Respiratory Disease. 126, (2), 332-337 (1982).
Maniecki, M. B., Møller, H. J., Moestrup, S. K., Møller, B. K. CD163 positive subsets of blood dendritic cells: The scavenging macrophage receptors CD163 and CD91 are coexpressed on human dendritic cells and monocytes. Immunobiology. 211, (6), 407-417 (2006).
Chignard, M., Balloy, V. Neutrophil recruitment and increased permeability during acute lung injury induced by lipopolysaccharide. American Journal of Physiology-Lung Cellular and Molecular Physiology. 279, (6), 1083-1090 (2000).
Coyne, C. B., et al. Regulation of Airway Tight Junctions by Proinflammatory Cytokines. Molecular Biology of the Cell. 13, (9), 3218-3234 (2002).
Durantie, E., et al. Biodistribution of single and aggregated gold nanoparticles exposed to the human lung epithelial tissue barrier at the air-liquid interface. Particle and Fibre Toxicology. 14, (49), (2017).
https://www.jove.com/t/61090
PDF DOI DOWNLOAD MATERIALS LIST
Barosova, H., Drasler, B., Petri-Fink, A., Rothen-Rutishauser, B. Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment. J. Vis. Exp. (159), e61090, doi:10.3791/61090 (2020).… More
Barosova, H., Drasler, B., Petri-Fink, A., Rothen-Rutishauser, B. Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment. J. Vis. Exp. (159), e61090, doi:10.3791/61090 (2020).
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text:
Mr. Antonym
Mr. Antonym is the moniker I have used to brand my solo electronic music output.
The Necronomicron Variations by Mr. Antonym
After more than fifteen years of not producing any significant new musical output, I happened to find myself stuck at home during what should have been a perfectly reasonable Christmas vacation.
It was the Omicron variant of COVID-19 that did it. It was a mild infection, but my vacation was ruined, since I had to quarantine myself at home.
Rather than let it go to waste, I took the opportunity to produce a brand new Mr. Antonym EP, The Necronomicron Variations. Three songs, made over the course of three days. I took about an additional week to make improvements, take care of details, and do the final mixing and mastering, and it was officially released on January 1, 2022. Available now from all your favorite music streaming services.
Mr. Antonym by Mr. Antonym
Since the late 1990s, one of my hobbies has been making electronic music.
My first solo album, available here for free download, represents some of the output of that hobby.
Here's some nice things that Randy Garcia of the Nophi Music Company (the label upon which it was originally released) said about it:
Mr. Antonym is a record we tried to release in 2006, but rescheduled at the opportunity to release a well-known artist that never fully materialized. We then promptly imploded for the first time.
The long and short: We were dumb to let this great collection of industrialized hip-hop beats crushing over sci-fi atmospherics and mangled synthesis go unheard. Our bad. It's still dope AF after 10 years in the vault.
– Randy Garcia
A live show in Orlando, Florida, circa 2007


Booking.com: Hotels in Bovec. Book your hotel now!
Located in Kranjska Gora, 5 miles from Podkoren Chairlift, Hiša Katja has accommodations with free WiFi, mountain views, a garden, and access to a spa center and a hot tub.
Life Hostel Slovenia is located in Radovljica, 25 mi from Ljubljana. There is a restaurant on site and guests can enjoy the on-site bar. Some rooms feature views of the mountain or a city park.
Linhart Hotel in Radovljica features an on-site coffee house with a terrace. Lake Bled is 4.3 mi away. Free WiFi is available.
Located in Dovje in the Gorenjska region, Mountain view Glamping features a terrace with views of the nearby mountains. The accommodations has free WiFi throughout the property.
Featuring barbecue facilities, a garden, and a terrace, Guesthouse ANKL has accommodations in Lesce with free WiFi and mountain views. Guests staying at this apartment can use the balcony.
Offering a terrace and free WiFi, House DorMica provides accommodations in Soča. All units are equipped with a TV and a fully equipped kitchen with a a fridge and coffee machine.
Located in Ribno in the Gorenjska region, Apartma Amelie features a garden. Guests might enjoy the free WiFi.
Located in Gozd Martuljek in the Gorenjska region, Apartma Jezerci has a garden. Guests can enjoy free WiFi. Opening onto a balcony, this air-conditioned apartment comes with 1 separate bedroom.
1,641 properties found in Bohinjska Bistrica ().


The counsellor could, in this case, be perceived as critical or at a distance. It is up to the counsellor to be transparent, congruent and honest about what is happening within the session. You may find that your client mentions the silence and this in itself, can be explored - the feelings around.... Different mindsets and emotional states require a particular approach; and the counsellor's ability to adjust to a client's needs normally dictates the outcome of that relationship. Also, within a counselling environment, the need may arise for a counsellor to work with clients who appear resistant to change or unhappy with external assistance.
UK World Europe IT is exactly two years since I decided I wanted to be a marriage guidance counsellor. My plan was to combine the counselling with my career as a freelance journalist, hoping... Mort Fertel is a world authority on the psychology of relationships and has an international reputation for saving marriages. In addition to working with couples, he teaches individuals how to single-handedly transform their marital situation.
The counsellor could, in this case, be perceived as critical or at a distance. It is up to the counsellor to be transparent, congruent and honest about what is happening within the session. You may find that your client mentions the silence and this in itself, can be explored - the feelings around.
This could be because of relationship difficulties, grief or to help them deal with everyday life. This page has information on the role of a counsellor, the entry requirements, skills needed and how to apply.
We can't find any apprenticeship vacancies in England for a Pet behaviour counsellor right now. The Find an apprenticeship service can help you with your search, send alerts when new apprenticeships become available and has advice on how to apply.


Single mature, reliable and caring woman available for short and long term sittings.
Experienced house and pets sitter. I am an animal and nature lover. I have owned dogs, cats, birds and chooks in the past. Minding them now is welcomed. I have pet minded a lizard or two in the past and also fish including Oscars and Siamese Fighting fish :) I am a keen gardener as well.
My most recent previous house sits have been in Alice Springs, NT.
I am a non-smoker, vegetarian, fit and healthy. I am an entreprenuer, travelling between Qld and NT for spiritual and cultural journeys that I offer throughout the year.
I have been a home owner and totally understand the need to have someone trustworthy and caring looking after your home, garden and pets.
I would love the opportunity to care for your house and pets. Please feel free to connect.


This year digital transformation and agile were the dominant themes along with the need to work in global timezones (hint, that makes the year even bigger whether you like it or not, it messes with your ability to be 'at rest').
I worked with global engineers, technology recruiting, state public servants, e-commerce businesses, and global agri business. Variety remained core to the client base, if not the type of work. And such very good people to work with – I am blessed.
I scaled down the one-on-one coaching and launched a new group webinar coaching product.
I created a new Agile leadership program with fellow Change Agent World Wide member Simon Terry.
I reinvigorated the Conversations of Change Retreats and piloted the new format.
I maintained the podcasts and watched the downloads and subscribers grow amazingly. Crikey!
Lena Ross and I also continued our #brainpickers series on youtube.
I partnered with the very fabulous BuzzPop Social to ensure that my social feeds were quality and engaging – if you think you saw more of me this year, it was the good work of Kate Ware, I reserved my time for engaging with people on social media, not posting or content creation. Where can I automate?" remained an enabling question all year round. I had very mixed experiences with outsourcing and delegating this year – I did more of it, it was hard work, and sometimes really dissapointing. It gave me an appreciation of why the clients I work with appreciate my attention to delivery and managing expectations. And also some good lessons in how to outsource and delegate better in future years because trust me, I made some really dumb mistakes.
I was an industry lecturer for Code Australia and an alumni facilitator for Daryl Conner in his 2017 Raising Your Game program.
I spoke with the folks from Able and How for OCtober, Trevor Young on my podcast, Des Walsh on his podcast, Elise Stevens on her podcast, Change Management Review on social media, and also the CMO Magazine on how Marketers need to embrace change.
And I launched my book. Oh yes, The Book. Where in one night we out trended the Bachelor's Australian launch on twitter!! What a trip!! People have asked me what is the hardest bit about writing a book – and it is by far the production and publicising it. A huge thank you to everyone who has supported me in this endeavour, I can't tell you how much I appreciate what you have done and especially those of you who have contacted me with feedback about the book. Sales have far surpassed what is standard for self published books and for that I couldn't be more grateful. It has been enthralling, confronting, confusing, and rewarding all wrapped up in one bundle.
Whether I identify with the role of educator or not, that's how many of my community see me. There is not always an appetite to pay for this knowledge though. I need to work through the commercial implications of this.
Building change capability at a practitioner level is not going to shift the dial as quickly and significantly as building change maturity with change leaders – I need to work with a greater volume of business leaders to have greater impact on how organisations deal with change.
I love speaking – this energises me, I'm really good at it, I need to do more.
I'm really good at developing new products. Expect more.
Currently, I'm probably quite confusing to many – they experience me professionally in many and varied forms. I'm not sure this is helpful to me, or others so I'll be seeking to reduce that confusion.
And what will the theme of 2018 be?
When I re-read what I have written above and acknowledge the swirling joyous messiness that was 2017, I don't know I want to change all that much. Simplify, automate, streamline, but not change the variety. Which means I need to ensure that I am well resourced internally to ride this crazy trip. And for me that means Serenity. Cultivating inner peace. Flow.
If with every opportunity, every engagement, every task I take on I do a serenity check first – will this bring me serenity / peace / flow or how do I ensure that I maintain a state of serenity / peace / flow, I'll end the year in pretty good shape. What about you?
Thank you for a wonderful year and for the experience of a couple of successful shared projects. Congratulations on all you achieved and for the insight of these great reflections. My only suggestion is don't let other people's desire for simplicity and certainty limit your ambitions or amazingly diverse talents. Some times the world (& the work of change agents) just has to be confusing to others.


text:
Arnold Walfisz (* 2. Juli 1892 in Warschau; † 29. Mai 1962 in Tiflis) war ein polnischer Mathematiker. Seine Schwerpunkte waren die Zahlentheorie und Algebra.

Leben
Nach dem Abitur in Warschau studierte er von 1909 bis 1914, sowie von 1918 bis 1921, in München, Berlin, Heidelberg und Göttingen, wo er 1921 bei Edmund Landau promovierte (Über die summatorischen Funktionen einiger Dirichletscher Reihen, 56 Seiten). Von 1922 bis 1927 lebte Walfisz in Wiesbaden. 1927 kehrte er nach Warschau zurück, wurde wissenschaftlicher Mitarbeiter an der Universität (ab der Habilitation 1930 als Privatdozent) und arbeitete, da diese Tätigkeit unbezahlt war, für die Versicherungsgesellschaft "Europa". 1935 gründete er mit Salomon Lubelski die Zeitschrift Acta Arithmetica. 1936 ging er nach Tiflis und wurde Mathematikprofessor an der dortigen Universität. Walfisz ist Autor von ungefähr 100 mathematischen Arbeiten, darunter die unter "Werke" genannten Bücher. Er hat auch zwei Bücher mit Schriften von Edmund Landau herausgegeben.

1936 bewies er den Satz von Siegel-Walfisz.

Walfisz nahm auch an Schachturnieren teil (2./3. Platz 1912 im Nebenturnier A in Breslau, Teilnahme 1914 beim nicht zu Ende gespielten Turnier in Mannheim).

Werke
Die Pellsche Gleichung (russisches Original: Уравнение Пелля), Tiflis, 1952
Gitterpunkte in mehrdimensionalen Kugeln, Panstwowe Wydawnictwo Naukowe, Monografi Matematyczne, vol. 33. Warszawa, 1957, online
Weylsche Exponentialsummen in der neueren Zahlentheorie, VEB Deutscher Verlag der Wissenschaften, Berlin, 1963

Literatur
 Ekkehard Krätzel, Christoph Lamm: Von Wiesbaden nach Tiflis – Die wechselvolle Lebensgeschichte des Zahlentheoretikers Arnold Walfisz, Mitteilungen der Deutschen Mathematiker-Vereinigung, Band 21 (1), 2013, S. 42–51,
 Sophie Goetzel-Leviathan (geb. Walfisz): Der Krieg von Innen, Paul Lazarus Stiftung, Wiesbaden, 2011
 A. J. Gillam: Mannheim 1914 and the Interned Russians, The Chess Player, Nottingham, 2014
 Paul Schellenberg u. a. (Hrsg.): Der 16., 17. und 18. Kongreß des Deutschen Schachbundes. Düsseldorf 1908, Hamburg 1910, Breslau 1912, 1984 (Buchreihe Tschaturanga)

Weblinks
Kurzbiographie bei der DMV
tabellarische Biografie und Schriftenverzeichnis
Kurzbiografie mit Bild aus Acta Arithmetica, 1964 (PDF-Datei; 4,23 MB)
Überblick seiner Arbeit aus Acta Arithmetica, 1964 (PDF-Datei; 2,31 MB)
Eintrag im Mathematics Genealogy Project

Einzelnachweise

Mathematiker (20. Jahrhundert)
Person (Warschau)
Hochschullehrer (Staatliche Universität Tiflis)
Pole
Geboren 1892
Gestorben 1962
Mann
Absolvent der Georg-August-Universität Göttingen

redpajama_set_name: RedPajamaWikipedia


Deadline 27 September! The ESRC and the Arts and Humanities Research Council (AHRC) are inviting applications for the UK-Japan Social Sciences, Arts and Humanities Connections grants, aimed at fostering the development of long-term relationships with Japanese researchers in the field of social sciences and humanities (SSH). The overall budget for this call is £1.5 million, with funding up to £50,000 (at 100% non standard fEC) per applicant for a maximum period of 18 months. More info and apply at http://bit.ly/2vvdPgs.
Closing 5 November! The John Crook Scholarship at St John's College, Cambridge is a fully funded scholarship to enable students who are the first in their family to go to university to study a second degree, either a 1 year Masters/MPhil or a 2 year BA Affiliated Degree. Find out more at http://bit.ly/2nrGNJV.
Meanwhile, over at the University of Lincoln, The School of Pharmacy is delighted (it says here – ed.) to offer four Fee Waiver scholarships (UK/EU only) for its Masters of Science (MSc) by Research Pharmacy and Pharmaceutical Sciences. This is a fantastic opportunity for postgraduate progression while tuition fees are covered by the School of Pharmacy.
…and this is just a taster of the stuff that'll be coming up as the new academic year gets into gear. Follow the blog and you won't miss out.
« Want to make a difference to the lives of thousands of children and families?


text:
Reviews of Attainable Hi-Fi & Home-Theater Equipment
Network Hub
Regular Reviews (up to 2011)
Shannon's Select Sounds (up to 2011)
SoundStage! Network (portal)
SoundStage! Access (here)
SoundStage! TV
Most Popular Featured Releases
Bobby Hutcherson: "Happenings"
War Poets: "Dulce et Decorum Est"
The Rolling Stones: "Exile on Main Street: Deluxe Edition"
Dusty Springfield, Small Faces, Herman's Hermits, Gerry and the Pacemakers: "British Invasion"
Miles Davis: "Miles! Live at Montreux: The Definitive Miles Davis at Montreux DVD Collection 1973-1991"
Mark Knopfler: "Down the Road Wherever"
Written by Joseph Taylor
British Grove/Blue Note B002920602
Musical Performance
Overall Enjoyment
It's not unusual for me to buy a new Mark Knopfler album, enjoy it for a few plays, then file it away, thinking, "Another solid album." I do the same with albums by Van Morrison and James Taylor and Joni Mitchell. But invariably, when I pull any of them off the shelf six months or so later, I find that I enjoy them even more than I remembered. Consistency can make me take certain musicians for granted, only to be reminded, when I play their music again, that their greatness remains undiminished.
Down the Road Wherever is Knopfler's ninth non-soundtrack solo album, and its first track, "Trapper Man," opens with a keyboard line from Guy Fletcher before Ian Thomas's drums hit hard to bring in Knopfler's familiar, softly distorted guitar. Knopfler tells the story of a trapper, "filth and grease on his clothes and hands," who brings his wares to a trader. The trapper reminds the trader, who wears "gems and rings," that it's the trapper's dirty work that makes possible his own good life. Knopfler fills the song with unrushed guitar lines that help set the tone for his story.
"Back on the Dance Floor" is as close to a return to the sound of Dire Straits as Knopfler has gotten in his solo career, and its dirty blues is a welcome sound. Imelda May's backing vocal gives the song a lift, but it's Guy Fletcher's keyboard flourishes and Glenn Worf's solidly funky bass that drive this track. Knopfler is a storyteller, and his guitar solos help establish the background of his narratives instead of being excuses to merely show off. His slide solo in "Just a Boy Away from Home" glides from the blues into a long quote from Rodgers and Hammerstein's "You'll Never Walk Alone" that gives this performance emotional focus.
Knopfler ventures into new territory with the easy funkiness of "Nobody Does That," which has a terrific horn chart reminiscent of the Average White Band and a gritty sax solo by Nigel Hitchcock. Tom Walsh's muted trumpet intro to "When You Leave" leads to some romantic crooning from Knopfler, whose voice continues to grow more fluid and expressive with age. The calypso soul of "Heavy Up" is also aided by a strong horn arrangement, and a deft, understated vocal from Knopfler.
Knopfler is adept at spinning tales, and two of the strongest on Down the Road Wherever are also the album's most personal. "One Song at a Time," from which the album gets its title, contains memories of Knopfler's travels as a musician, absorbing history and experiences to use in his songs. "Matchstick Man" grew out of a memory of hitchhiking home after a gig -- with only his voice and acoustic guitar, Knopfler paints a picture of a young musician making his way in the world.
Many of the musicians on Down the Road Wherever have often worked with Knopfler before; there's an ease and professionalism in the sound of this album that, on first hearing, might feel too laid-back. But repeated listenings reveal more depth in the arrangements, more subtle interplay among the players. And Knopfler always uses his considerable guitar skills in service to his songs. The quote from "You'll Never Walk Alone" first struck me as sentimental, but after I'd listened a few more times, I found that it underlined and strengthened the feeling of hope contained in the song's closing lines.
Knopfler and Fletcher coproduced and recorded the album at Knopfler's British Grove Studio, which prides itself on using a mixture of old and new audio technologies. The sound is clean and expansive, with a broad soundstage that provides plenty of breathing room for all of the instruments and voices. At this point Mark Knopfler has nothing to prove, but Down the Road Wherever is proof that he's not coasting -- it's another impressive entry in a rewarding discography.
. . . Joseph Taylor
josepht@soundstagenetwork.com
SoundStage! Access (formerly GoodSound!) is part of
This site was designed by JoomlaShack, Karen Fanas, and The SoundStage! Network.
Visit NADElectronics.com


text:
Sir John Maxwell
Burrell Collection Photo Library
A portrait of Sir John Maxwell 2nd Baronet (1686-1752) by an unknown artist.
Sir John Maxwell of Blawarthill inherited Nether Pollok from his cousin Sir John Maxwell (1648-1732) in 1732. The second baronet kept up with fashions of his day, building a modern mansion, Pollok House, and sending his son on a Grand Tour of Europe. He also encouraged industrial developments on his estates and granted a feu for a printfield, stimulating the development of Pollokshaws as an industrial village.
Sir John married three times, and portraits of his first two wives Anne Carmichael (d 1720) and Barbara Stewart (d 1737) are also on this website.
Reference: PC 180
Reproduced with the permission of John Maxwell Macdonald
fashions, oil paintings, Pollok Estate, Pollok House, Pollok Park, portraits, printfields, wigs


text:
var testCafeAutomation = window.getTestCafeModule('testCafeAutomation');
var RClickAutomation   = testCafeAutomation.RClick;
var ClickOptions       = testCafeAutomation.get('../../test-run/commands/options').ClickOptions;

var testCafeCore = window.getTestCafeModule('testCafeCore');

testCafeCore.preventRealEvents();

var hammerhead   = window.getTestCafeModule('hammerhead');
var browserUtils = hammerhead.utils.browser;

var RIGHT_BUTTON_WHICH_PARAMETER = hammerhead.utils.event.WHICH_PARAMETER.rightButton;

$(document).ready(function () {
    //constants
    var TEST_ELEMENT_CLASS = 'testElement';

    //utils
    var addInputElement = function (type, id, x, y) {
        var elementString = ['<input type="', type, '" id="', id, '" value="', id, '" />'].join('');

        return $(elementString)
            .css({
                position:   'absolute',
                marginLeft: x + 'px',
                marginTop:  y + 'px'
            })
            .addClass(type)
            .addClass(TEST_ELEMENT_CLASS)
            .appendTo('body');
    };

    var startNext = function () {
        if (browserUtils.isIE) {
            removeTestElements();
            window.setTimeout(start, 30);
        }
        else
            start();
    };

    var removeTestElements = function () {
        $('.' + TEST_ELEMENT_CLASS).remove();
    };

    //tests
    QUnit.testDone(function () {
        if (!browserUtils.isIE)
            removeTestElements();
    });

    module('dom events tests');

    asyncTest('mouse events raised', function () {
        var $input            = null;
        var mousedownRaised   = false;
        var mouseupRaised     = false;
        var clickRaised       = false;
        var contextmenuRaised = false;

        $input = addInputElement('button', 'button1', Math.floor(Math.random() * 100),
            Math.floor(Math.random() * 100));

        window.setTimeout(function () {
            $input.mousedown(function (e) {
                mousedownRaised = true;
                ok(e.which, RIGHT_BUTTON_WHICH_PARAMETER);
                ok(!mouseupRaised && !clickRaised && !contextmenuRaised, 'mousedown event was raised first');
            });
            $input.mouseup(function (e) {
                mouseupRaised = true;
                ok(e.which, RIGHT_BUTTON_WHICH_PARAMETER);
                ok(mousedownRaised && !clickRaised && !contextmenuRaised, 'mouseup event was raised second');
            });
            $input.click(function () {
                clickRaised = true;
            });
            $input.contextmenu(function (e) {
                contextmenuRaised = true;
                ok(e.which, RIGHT_BUTTON_WHICH_PARAMETER);
                ok(mousedownRaised && mouseupRaised && !clickRaised, 'contextmenu event was raised third ');
            });

            var rclick = new RClickAutomation($input[0], new ClickOptions({ offsetX: 5, offsetY: 5 }));

            rclick
                .run()
                .then(function () {
                    ok(mousedownRaised && mousedownRaised && !clickRaised && contextmenuRaised, 'mouse events were raised');
                    startNext();
                });
        }, 200);
    });

    asyncTest('T191183 - pointer event properties are fixed', function () {
        var mousedownRaised = false;
        var mouseupRaised   = false;
        var contextmenu     = false;

        var $el = addInputElement('button', 'button1', Math.floor(Math.random() * 100),
            Math.floor(Math.random() * 100));

        $el.mousedown(function (e) {
            mousedownRaised = true;

            equal(e.button, 2);

            if (!browserUtils.isSafari)
                equal(e.buttons, 2);

            ok(!mouseupRaised && !contextmenu, 'mousedown event was raised first');
        });

        $el.mouseup(function (e) {
            mouseupRaised = true;

            equal(e.button, 2);

            if (!browserUtils.isSafari)
                equal(e.buttons, 0);

            ok(mousedownRaised && !contextmenu, 'mouseup event was raised second');
        });

        $el.contextmenu(function (e) {
            contextmenu = true;

            equal(e.button, 2);

            if (!browserUtils.isSafari)
                equal(e.buttons, 0);

            ok(mousedownRaised && mouseupRaised, 'click event was raised third ');
        });

        var pointerHandler = function (e) {
            equal(e.pointerType, browserUtils.version > 10 ? 'mouse' : 4);

            if (e.type === 'pointerdown')
                equal(e.buttons, 2);

            if (e.type === 'pointerup')
                equal(e.buttons, 0);
        };

        if (browserUtils.isIE && browserUtils.version > 11) {
            $el[0].onpointerdown = pointerHandler;
            $el[0].onpointerup   = pointerHandler;
        }
        else {
            $el[0].onmspointerdown = pointerHandler;
            $el[0].onmspointerup   = pointerHandler;
        }

        var rclick = new RClickAutomation($el[0], new ClickOptions({ offsetX: 5, offsetY: 5 }));

        rclick
            .run()
            .then(function () {
                ok(mousedownRaised && mousedownRaised && contextmenu, 'mouse events were raised');

                if (browserUtils.isIE)
                    expect(14);
                else if (browserUtils.isSafari)
                    expect(7);
                else
                    expect(10);

                startNext();
            });
    });
});

redpajama_set_name: RedPajamaGithub


Determination to "Turn our wills and our lives and our game over to the care of God as we understand Him".
We need a strong foundation. We cannot find it in mere positive thinking or visualization.
The "secret" doesn't live in the "SECRET". If you are trying all those mental high jinks you will find that they won't work alone without a spiritual core.
We may believe in a God, but may have not let Him into our lives. If we are determined to find God's will for us, we will be rewarded. We must become determined to live on purpose.
The success of any 12 step program rests on how determined we are to follow this step.
Even the most modest attempts of letting go and letting God will bear fruit. The door of faith will open wider as we become more willing to trust God. Self will always closes it.
The more determined we become to turn things over to our Higher Power, the more we become more independent. People, places and things which held us captive are no longer our masters. We gain true spiritual freedom.
We have in the past determined our intellect and self will would find us inner peace and grant us the success we sought.
Everywhere we look, we see people filled with anger and fear and despair. We have looked to celebrity as the benchmark of our own self worth and self image.
We see the fragmentation of our countries, our communities and our families. We have become anarchists living for the self. We seek to have the thirst of the moment quenched.
The effect of our living outside consciousness is to live in the dark. We are sleep walkers. We have become automatons chasing the "flavor of the month", we have no foundation.
We were given the gift of free will, and we believe we exercise that will freely. To execute our free will, we have to become awake. Become determined to achieve awareness.
Hedonism seems too strong a word for the way we function, but is it? Hedonism is the belief which holds that the good is whatever gives you pleasure and, ergo pleasure is the standard of morality. So as our pleasures change so does our basis for morality, guided by our feelings, emotions and whims and not by our minds.
A. for every action there is a reaction.
We are determined to lay down our desires to play God. We have admitted defeat, have begun to believe in a Higher Power, and now we make a decision to turn our will and our lives over to His care.
Far from being a weakness, this dependence becomes our strength. When we begin to align our will with that of God's, we begin to use our will freely.
"Our whole trouble has been the misuse of will power. We have tried to bombard our problems with it instead of attempting to bring it into agreement with God's intention for us". Bill W.


Having trouble with your rear liftgate staying up on your Jeep Wrangler Grand Cherokee. Tired of loading groceries with one hand holding up the liftgate. Time to replace your worn out Liftgate Strut with a new factory style replacement by Crown Automotive. Mounts to the original location using the factory hardware. Passenger (right) side only for power liftgate only. Offering a 12 Month/12,000 Mile Limited Warranty.
Original Replacement: Designed as a direct replacement for any easy installation.
Quality Product: Custom made for your 11-13 Grand Cherokee WK2 with power liftgate and made to factory specifications.


This post regarding Named Pilots is part of a larger article regarding Wave 1 listbuilding- the rest of the posts n this series will be available in the coming weeks as I'm revealing a post or two per day on the various topics in that larger article. For more info, click the "Ship List Articles" tab at the top of the page or simply click here.
Card: Backstabber (TIE Fighter) 16 Pts.
Card Text: When attacking outside the defender's' firing arc, roll 1 additional attack die.
Ability is an Action?: No.
Quick Take: Most folks run TIEs straight at their opponents to get into Range 1 to help compensate for only having 2 attack dice. While Backstabber can do this, and does it very well getting a massive 4 attack dice if still managing to stay out of his target's firing arc at Range 1, I think perhaps a more important use for Backstabber is to provide some more long range fire support- something missing in the Wave 1 Imperial arsenal. For whatever reason, a lot of folks don't seem to realize that Backstabber's ability works regardless of range, so from an attacking perspective, he's more or less an X-Wing so long as he's outside his target's firing arc, which when you've got as many moves as a TIE fighter, shouldn't be that hard.
Flying Backstabber on flanks where he can more properly exploit his special ability is a great move. Pairing him up with Vader as the rovers accompanying a TIE pile supported by Howlrunner is the basis of a great Imp swarm list.


Kesh Kala Super Vasmol 33 Natural Black Hair Darkening Cream The safest way to beautiful black hair Emulsion Type Hair Dye PPD content not more than 4% No Ammonia No Peroxide No Mixing Super Vasmol 33 is unlike any other hair dye. A unique, innovative, easy to use, Super Vasmol 33, covers grey hair making them naturally black in just a single application. It contains No Ammonia or Peroxide. Now darkens hair the natural way. It contains Ayurprash, a combination of various beneficial herbal extracts which are known to strengthen hair roots, impart shine. Super Vasmol 33 contains Tea Tree Oil having Anti Dandruff properties. - Keep the product out of reach of children - Protect from heat & direct sunlight. - For external use only.


Fresh strawberries, sugar and fruit pectin are cooked briefly then processed in a canner for scrumptious homemade jam.
Stem and crush strawberries thoroughly, one layer at a time. Measure exactly 5 cups prepared fruit into 6- or 8-qt. saucepot.
Stir pectin into fruit in saucepot. Add butter to reduce foaming. Bring mixture to full rolling boil (a boil that doesn't stop bubbling when stirred) on high heat, stirring constantly. Stir in sugar. Return to full rolling boil and boil exactly 1 min., stirring constantly. Remove from heat. Skim off any foam with metal spoon.


Mark's favourite fruit- Mangos are in full season. At the moment we are spoiled, Scott's mum and dad who live up north in tropical Queensland have a grove of Bowen Mango trees, and Mark gets a box or two of newspaper wrapped fruit every summer. Our new place has a large Common Mango tree, the fruit are not quite as good as the Bowen variety, but the tree is laden with fruit that are just turning colour and ripening. One of the bad things about having a mango tree in your back yard is fruit bats, and we have a large colony of them. Bats are a real nuiscance. They poop on your washing and half eat the fruit on the tree. Nothing is funnier then Mark standing under the tree telling them to piss off and leave his mangos alone, and to then hear a half-eaten mango plop to the ground.
1 cup all-purpose flour (or rice flour) mixed with 1/2 tsp.
Place all 'Mango Sauce' ingredients in a food processor or blender. Process well, until more or less smooth.
Taste-test the sauce. The goal is to reach a balance of sweet, sour, spicy, and salty. Add more sugar if you find the sauce too sour (this will depend on the sweetness of your mangoes). If not spicy enough, add more chili. If not salty enough, add more fish sauce. If too salty or too sweet, add more lime juice. Set aside.
Stir the flour and salt together and place in a mixing-type bowl. Add the chicken pieces and turn or gently stir them to coat.
Wash out the wok/frying pan, or use another (clean) one. Set over medium-high heat and add the mango sauce plus red pepper. Bring to a gentle boil, then reduce to a simmer (medium to low heat). If sauce becomes too thick, add 3-4 Tbsp. water. Simmer 3-4 minutes, or until pepper has softened slightly but still retains some crispness.
Add the fried chicken pieces, gently stirring them in to cover with sauce. If desired, add chunks of mango (fruit of 1/2 to 1 ripe mango) too. Simmer briefly, just until everything is hot.
Do a final taste-test for salt (tasting the sauce together with the chicken). Add a little more fish sauce or chili if desired.