Isolation and immunofluorescence staining of Aspergillus fumigatus conidia-containing phagolysosomes

Summary The analysis of phagolysosomes within professional phagocytic cells is facilitated by their isolation. Here, we optimized a protocol for the isolation of intact phagolysosomes from macrophages infected with the spores of Aspergillus fumigatus. Purified phagolysosomes allow improved immunostaining, e.g., of phagolysosomal membrane proteins, or proteome analysis. For complete details on the use and execution of this protocol, please refer to Schmidt et al. (2020).


SUMMARY
The analysis of phagolysosomes within professional phagocytic cells is facilitated by their isolation. Here, we optimized a protocol for the isolation of intact phagolysosomes from macrophages infected with the spores of Aspergillus fumigatus. Purified phagolysosomes allow improved immunostaining, e.g., of phagolysosomal membrane proteins, or proteome analysis. For complete details on the use and execution of this protocol, please refer to Schmidt et al. (2020).

BEFORE YOU BEGIN
The protocol is optimized from the published work of Kyrmizi et al. (2018).
1. Phagocytic cells should be maintained according to the accepted protocols. Their viability should be higher than 90%, and confluency in flasks or wells should be around 80%. 2. For efficient uptake by phagocytic cells, fungal spores should be freshly harvested from mycelia.

MATERIALS AND EQUIPMENT
For cultivation of RAW264.7 macrophages, the listed DMEM medium is recommended.

STEP-BY-STEP METHOD DETAILS
Preparation of A. fumigatus conidia and macrophage cell line (RAW264.7) For the isolation of phagolysosomes from macrophages seeding of a defined amount of macrophages in well plates and cultivation of A. fumigatus conidia until sporulation is necessary and described below.
Timing: 10 min 1. Revival of A. fumigatus conidia from cryopreservation a. Use an inoculation loop to spread conidia on a petri dish with Aspergillus Minimal Medium agar (Pontecorvo et al., 1953) b. Incubate conidia-plated agar plate for 5 days at 37 C Timing: 30 min plus 3 days 2. Seed macrophage cell line a. Preheat cell culture medium (see ''Materials and equipment''), which should be stored at 4 C, to 37 C in a water bath b. Prepare 2 3 25 cm 2 cell culture flasks with 3 3 10 5 macrophages (revived from cryopreserved cell line) and 7 mL culture medium each c. Incubate flasks for 3 days at 37 C and 5% (v/v) CO 2 Timing: 1 h plus 1 day 3. Harvest conidia 5 days after mycelial growth and sporulation on agar plates a. Slowly add 10 mL 0.9% (w/v) NaCl, 0.01% (v/v) Tween 20 solution to the petri dish b. Use a T-formed spatula to scrap the conidia c. Measure concentration of conidia by using a 1:100 dilution and counting with a Thoma chamber or another counting method 4. Harvest adherent macrophages with a confluency of 80%-90% Infection of macrophages with A. fumigatus conidia Macrophages are incubated with a defined amount of conidia to allow phagocytosis and formation of a conidia-containing phagolysosome. The implementation of infection is explained in the following.
Timing: 3 h 6. Replace culture medium of the 4-well plate containing macrophages a. Remove the old medium b. Add 3 mL pre-warmed fresh medium to the cells 7. Phagocytosis of A. fumigatus conidia by macrophages a. Infect macrophages using an MOI of 5; incubate for 30 min at 4 C to synchronize phagocytosis b. Allow phagocytosis for 2 h at 37 C and 5% (v/v) CO 2 Isolation of conidia-containing phagolysosomes Formed phagolysosomes are isolated by the described cell lysis and Biocoll-separating process.
Timing a. Split the 1 mL homogenate into portions of 500 mL; carefully overlay each 500 mL on top of 500 mL Biocoll (1.077 g/mL) in a reaction tube by using a 200 mL tip CRITICAL: A small pipette tip avoids too fast layering of the homogenate onto Biocoll and improves separating process.
b. Centrifuge at 600 3 g for 20 min at 4 C c. Discard supernatant d. Wash pelleted conidia-containing phagolysosomes twice with ice-cold 500 mL PBS with centrifugation in-between for 5 min, 600 3 g, at 4 C e. Resuspend pellet in 150 mL ice-cold PBS and collect all samples in one tube f. Seed 20 mL suspension + 1 mL PBS or count phagolysosomes and place a certain amount on a coverslip in a 24-well plate and store at 4 C (possible for max. 1 day) to allow the phagolysosomes to attach onto the coverslip. Alternatively, isolated phagolysosomes can be lysed for Western Blot analysis.

Immunofluorescence staining of conidia-containing phagolysosomes
Timing: 6 h Membrane-associated targets of isolated phagolysosomes can be stained with specific antibodies using immunofluorescence technology as described below.

EXPECTED OUTCOMES
For each condition, you should prepare 2 wells of a 4-well plate to isolate a sufficient number of phagolysosomes for immunofluorescence. If 20 mL of phagolysosome suspension are applied to a glass slide of a 24-well plate, it is possible to analyze 7-8 wells per condition in total. An example of an immunofluorescence staining of the vATPase V 1 subunit on isolated phagolysosomes of RAW264.7 macrophages infected with A. fumigatus pksP conidia is shown in Figure 1.
The protocol can also be used for isolation of phagolysosomes from other cell lines and primary cells.
Its suitability was confirmed for J774A.1 macrophage cell line and primary bone marrow-derived monocytes/macrophages from C57BL/6 mice.

QUANTIFICATION AND STATISTICAL ANALYSIS
Samples were visualized using a Zeiss LSM 780 confocal microscope and images were processed with the Zeiss ZEN software. Phagolysosomes with a positive signal for a specific target were ll OPEN ACCESS counted and related to non-stained phagolysosomes. One hundred conidia-containing phagolysosomes per biological replicate were evaluated. The values represent the mean G SD of three biological replicates. Additionally, immunofluorescence intensities could also be evaluated bioinformatically (Schmidt et al., 2020).

LIMITATIONS
The protocol leads to highly purified phagolysosomes; however, some cell debris or intact macrophages may remain in the suspension. A distinction of phagolysosomes from cell debris or intact cells is feasible by microscopy. A quantification of exclusively phagolysosomal proteins by Western blot proved to be more difficult.

TROUBLESHOOTING Problem 1
The phagolysosomal suspension remains intact cells.

Potential solution
If effective isolation is uncertain, verification of damaged cells via trypan blue staining is necessary. Additional passing steps through a needle are recommended to further reduce the number of intact cells in the sample. Additionally, a careful overlaying improves phase separation between Biocoll and lysate. The use of a yellow tip for a pipetman facilitates the overlaying and improves the separation of phagolysosomes in the pellet and intact cells in the Biocoll-supernatant.

Problem 2
Phagolysosomes are not efficiently stained.

Potential solution
Successful staining of phagolysosomes with antibodies often depends on the fixation reagent, e.g., formaldehyde or methanol/acetone. If the immunofluorescence signal is weak or non-specific, other fixation methods or antibody concentrations can be assessed. For formaldehyde fixation, an addition of 0.3 M glycine to the blocking solution blocks free aldehyde groups and prevents non-specific binding. Problem 3 Isolation of phagolysosomes only allows immunofluorescence of target structures on the outer membrane of phagolysosomes.

Potential solution
If your target of interest is expected on the inner phagolysosomal membrane, a permeabilization step with 1% (v/v) Triton X-100/PBS is recommended.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Axel A. Brakhage (axel.brakhage@leibniz-hki.de).

Materials availability
Any material generated in this study is available from the Lead Contact with a completed Materials Transfer Agreement.

Data and code availability
This study did not generate any unique datasets or code.