In vitro analyses of paracrine effects of murine classically activated macrophage on beige adipocyte metabolism

Summary The communication between macrophage and adipocyte plays a critical role in the initiation and development of metabolic inflammation, which is difficult to study in vivo. Here, we provide a step-by-step protocol using differentiated cells to investigate the paracrine effects of classically activated macrophage on beige adipocyte metabolism in vitro. This protocol uses bone-marrow-derived macrophage and SVF-derived UCP1+ beige adipocyte in a culture model to study immune regulation of adipocyte metabolism by western blot analyses. For complete details on the use and execution of this protocol, please refer to Yao et al. (2021).


SUMMARY
The communication between macrophage and adipocyte plays a critical role in the initiation and development of metabolic inflammation, which is difficult to study in vivo. Here, we provide a step-by-step protocol using differentiated cells to investigate the paracrine effects of classically activated macrophage on beige adipocyte metabolism in vitro. This protocol uses bone-marrow-derived macrophage and SVF-derived UCP1 + beige adipocyte in a culture model to study immune regulation of adipocyte metabolism by western blot analyses. For complete details on the use and execution of this protocol, please refer to Yao et al. (2021).

BEFORE YOU BEGIN
Institutional permissions Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University and conformed to the Guide for the Care and Use of Laboratory Animals (IMM-QiuYF-1).
Preparation of mice CD-1(ICR) mice are used for the differentiation of bone marrow derived macrophage (BMDM), at the age of 8-12 weeks. CD-1(ICR) mice are used for stromal vascular fraction (SVF) isolation, at the age of 12-16 days.
Note: C57BL/6 mice can also be used for both BMDM and SVF isolation and differentiation.

STEP-BY-STEP METHOD DETAILS Preparation and culture of bone-marrow-derived macrophage
Timing: 6-7 days 1. Euthanize 8-12 weeks old CD-1 mice according to an approved IACUC protocol. 2. Isolate the femurs and tibias at joint, and remove skin and muscle as clear as possible. 3. Wash 3 times with PBS, then transfer to a new 10 cm dish with PBS at room temperature (RT: 18 C-24 C). 4. Cut off the ends of the bones and flush with PBS using a 23-gauge needle. Collect bone marrow in a 50 mL Falcon tube. 5. Centrifuge at 400 3 g for 5 min at RT (18 C-24 C) and aspirate supernatant.
CRITICAL: Do not centrifuge at cold condition because the density of Ficoll will change.
9. Collect all the supernatant (both Ficoll and BMDM medium, but not the pellet) into a 50 mL tube. 10. Add 25 mL pre-warmed PBS and mix well. 11. Centrifuge for 5 min at 200 3 g. 12. Resuspend the cell pellet in BMDM growth medium and plate into 2 Falcon dishes (15 cm).
CRITICAL: Do not use cell culture dish, which will make it difficult to digest differentiated BMDM.
CRITICAL: It is very important to change the medium at day 5 and 6 for the differentiation of BMDM. (Troubleshooting 1) 14. By day 7, cells should be fully differentiated and are ready to be plated. 15. Wash cells with 5 mL PBS once, and then incubate with 5 mL 5 mM EDTA (in PBS) for 5 min in cell incubator.
Note: Do not use trypsin for digestion, which will activate the macrophage, and the digestion should not be longer than 10 min. (Troubleshooting 3) 16. View cells under microscope to ensure that half of cells are detached. 17. Add another 5 mL pre-warmed BMDM growth medium to stop the digestion by EDTA and resuspend the cells. 18. Centrifuge for 5 min at 200 3 g. 19. Resuspend the cell pellet with 5 mL BMDM growth medium. 20. Count and adjust the final cell density to 4-6 3 10^5/mL. 21. Plate 1 mL cell suspension per well into a 12-well cell culture plate. 22. After 8-16 h, the plated mature macrophages are stimulated with PBS or 100 ng/mL lipopolysaccharide (LPS) for 4 h (Figure 1).
Optional: You can also polarize the BMDM to classically activated macrophage or alternatively activated macrophage by 10 ng/mL LPS or 10 ng/ml IL-4 treatment for 24-48 h.
23. Aspirate medium gently, then wash with pre-warmed PBS twice and add fresh BMDM growth medium. 24. After 12 h, the conditioned medium is collected for cell treatment or stocked at À80 C. 25. For extracting RNA of BMDM, add 350 mL TRIzol per well and transfer to 1.5 mL tubes. Then perform qRT-PCR to analyze proinflammatory gene expression in BMDM (Figure 2).
Preparation and culture of SVF-derived beige adipocyte Note: Make sure not to penetrate the peritoneal cavity.
30. Pull ventral skin away from both flanks gently and slowly. 31. Identify the scWAT depot, then use scissor to trace the edges of the tissue and cut along the way until the entire depot is detached from the skin (Figure 3). 32. Transfer dissected scWAT into pre-chilled scWAT digestion buffer in a 6 cm dish immediately. 33. Wash scWAT twice with pre-chilled scWAT digestion buffer. 34. Transfer the tissues to a 1.5 mL tube with 200 mL scWAT digestion buffer supplemented with 600 IU/mL collagenase type I. 35. Mince the tissues by using sterile surgical scissors. Tissue pieces should be $1-2 mm thick. 36. Transfer tissues and scWAT digestion buffer into a 50 mL sterile conical tube containing 15 mL scWAT digestion buffer supplemented with 600 IU/mL collagenase type I. 37. Place the tube horizontally and digest tissues at 37 C with shaking at 100 rpm for 40-60 min.
Note: Digestion time depends on disappearance of visible pieces of adipose tissue, but no more than 60 min.    (SVF cells from every mouse (two pieces of scWAT) resuspended by 2 mL medium). 47. Plate 1 mL cell suspension per well into a 12-well flat-bottom plate.
Note: The SVF cells from one mouse can be seeded to 2 wells of 12-well flat-bottom plate.
48. After 12-24 h, remove the beige adipocyte maintenance medium carefully and wash the cells with pre-warmed PBS for twice. 49. Change beige adipocyte maintenance medium every 2 days until the cells reach 90% confluent. 50. When the cells reach 90% confluent (day 0), remove beige adipocyte maintenance medium and add 1 mL beige adipocyte induction medium per well.
CRITICAL: Beige adipocyte induction medium has to be prepared freshly prior to usage.
51. On day 2, remove beige adipocyte induction medium and add 1 mL beige adipocyte differentiation medium per well.
CRITICAL: Beige adipocyte differentiation medium has to be prepared freshly prior to usage.
52. On day 4, remove the medium and add 1 mL fresh beige adipocyte differentiation medium per well. 53. Change beige adipocyte differentiation medium every day until the cells are fully differentiated to beige adipocyte on day 7-8 ( Figure 4).

BMDM conditioned medium treatment for western blot analysis
Timing: $24 h 54. Remove beige adipocyte differentiation medium of beige adipocyte and add 0.5 mL beige adipocyte maintenance medium per well. 55. Add 0.5 mL conditioned medium from PBS-or LPS-treated BMDM per well. 56. Culture at 37 C 5% CO 2 for 12 h. 57. Add vehicle (Veh) or 50 nM norepinephrine to differentiated beige adipocyte directly without changing the medium. 58. Culture at 37 C 5% CO 2 for 8 h. 59. Remove the medium and wash cells twice with pre-chilled PBS. 60. Remove as much of the PBS as possible. 61. Add 300 mL RIPA supplemented with protease inhibitor cocktail per well. 62. Resuspend the cells and transfer to 1.5 mL tubes. 63. Lyse the cells for 10 min on ice and then vortex. 64. Centrifuge for 20 min at 15,000 3 g and transfer 200 mL supernatant to 1.5 mL tubes.
Note: Avoid to aspirate or contact the sediment as carefully as possible. (Troubleshooting 5).

EXPECTED OUTCOMES
In preparation and culture of bone marrow-derived macrophage, it shows that many non-adherent cells detach from day 2-3, which are erythrocytes, neutrophils and other bone marrow-derived cells. They can be removed when changing the medium at day 3. The adherent cells will be very sparse before day 3 and increase rapidly from day 4-5. For bone marrow cell isolation from one male CD-1 mouse (2 femurs and 2 tibias), it can generate 5-10 3 10 7 macrophages at day 6-7. The In preparation and culture of SVF-derived beige adipocyte, about 12-24 h after plating, the confluence of preadipocytes from scWAT SVF should be 20%-50%. The cell confluence will increase to 90% after 1-3 days. After changing to beige adipocyte induction medium, cells will drastically alter their morphological appearance and lipid droplets will appear from day 3-4. Then, the differentiating cells will increase the number and size of lipid droplets as the differentiation process goes on. Eventually, the differentiation efficiency will be 70%-90% at day 7 ( Figure 4).
Proinflammatory cytokines, including IL-1b and TNF, play an important role in the inhibition of adipocyte lipolysis and thermogenesis (Goto et al., 2016;Mowers et al., 2013). Thus, the lipolysis and thermogenesis of beige adipocyte can be inhibited by the LPS-treated BMDM medium when compared with the PBS-treated ( Figure 5).

LIMITATIONS
Firstly, differentiated mature beige adipocytes are viable for only 2-4 days. Thus, the cells will float and fall off once they are cultured for more than 2 days. Secondly, BMDM and beige adipocyte generated using this protocol are useful for analyzing their common functions, but they cannot reflect the heterogeneity of cells in vivo. For example, there are different types of macrophage in different tissues, and even many different types in one tissue (Epelman et al., 2014;Felix et al., 2021;Lavin et al., 2015). Thirdly, these experiments cannot completely mimic the interactions between macrophage and beige adipocyte in vivo by using this protocol. In adipose tissue, macrophage can interact with beige adipocyte both directly and indirectly (Cohen and Kajimura, 2021). The treatment of beige adipocyte by conditioned BMDM medium can only mimic the effects of indirect interaction, but not the direct one via physical contact.

Potential solution
It can be expected to generate at least 5 3 10 7 macrophages from one mouse (2 femurs and 2 tibias). If the total cell number is less than 1 3 10 7 , the following improvements should be considered. Firstly, make sure that the mice are no more than 16 weeks old. The perfect age is from 8 to 12 weeks. Secondly, rinse the bone marrow out as much as possible, until the bone turns white. Thirdly, the BMDMs should be changed to fresh medium strictly every day after day 5.

Potential solution
Firstly, Penicillin-Streptomycin should be added into the BMDM growth medium to prevent bacterial contamination. Secondly, skin and muscle attached to bone should be removed as completely as possible. Thirdly, bone should be washed as least twice before flushing bone marrow.
Problem 3 BMDM is activated after digestion. (step 15 in ''preparation and culture of bone marrow-derived macrophage'')

Potential solution
The digestion time is very important, which should be limited strictly between 3-10 min. If it is longer than 10 min, the viability of BMDM will decrease. If it is shorter than 3 min, the BMDM has to be pipetted harder, which may cause cell damage and activation.

Problem 4
Low differentiation efficiency of beige adipocyte. (step 53 in ''preparation and culture of SVFderived beige adipocyte'')

Potential solution
Usually, the differentiation efficiency of beige adipocyte can reach over 80%. If it is less than 70%, the following improvements should be considered. Firstly, the beige adipocyte induction medium and differentiation medium should be prepared freshly before usage. Secondly, the cell confluence should be limited strictly between 90%-100% when changing to beige adipocyte induction medium. Thirdly, digestion time for scWAT tissue should not be too long and restrict the time to no more than 60 min. Fourthly, the mice used for scWAT SVF isolation should not be older than 16 days.

Problem 5
It is difficult to get pure protein samples for western blot analysis when extracted from beige adipocytes. (step 64 in ''BMDM conditioned medium treatment for Western Blot analysis'') Potential solution It is mainly due to an incomplete removal of lipids. The cell lysate of beige adipocytes contains a lot of lipids, which cannot be precipitated and thus spread in the supernatant or aggregate in the top as a low-density and insoluble layer. To make the supernatant purer, one should add more RIPA or centrifuge it for several additional times.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yifu Qiu (yifu.qiu@pku.edu.cn).

Materials availability
This study did not generate new unique reagents.

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