C57BL/6 wild-type mice were euthanized by cervical dislocation, followed by disinfection through immersion in 75% alcohol.
The thorax was incised along the midclavicular line bilaterally, exposing the white thymic tissue located posterior to the sternum.
The thymic tissue was rinsed with PBS and subsequently placed on a 40μM mesh for grinding. The resultant cell suspension was collected in a centrifuge tube.
Red blood cells were eliminated using a red blood cell lysing solution.
After PBS washing, the cells were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).
Apoptosis was triggered by incubating the primary murine thymocytes with 50nM dexamethasone for 3 hrs.
CFSE staining
Thymic primary cells were stained with CFSE according to the manufacturer's instructions. The staining process was conducted under light-protected conditions.
Efferocytosis assay
RAW264.7 or bone marrow-derived macrophage (BMDM) cells were cultured in a 6-well plate (1×10^6 cells/well) with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum.
CFSE-labeled apoptotic thymocytes were added at a 10:1 ratio to the macrophage cultures and incubated for an additional 120 minutes.
Cells were thoroughly washed three times with PBS to remove non-engulfed thymocytes.
For flow cytometry, cells remaining in the culture plate were detached and resuspended for analysis. Flow cytometry was performed to determine the proportion of CFSE-positive cells in relation to the total cell count.
For immunofluorescent staining, macrophages were plated on cell climbing tablets in a 6-well or 12-well plate. After removal of excess thymocytes following phagocytosis, cells were fixed using 4% paraformaldehyde.
Following immunofluorescence staining and mounting with DAPI, samples were visualized using a fluorescence microscope.
For statistical analysis, each glass slide is randomly selected for 5-10 fields of view at 400X magnification. Efferocytosis index were calculated using the following formula: (number of macrophages containing apoptotic bodies)/(total macrophages) × 100%. The index was then normalized using the control group as the reference point (100%).
Yao, Z., Qi, W., Zhang, H., Zhang, Z., Liu, L., Shao, Y., Zeng, H., Yin, J., Pan, H., Guo, X., Liu, A., Cai, D., Bai, X. and Zhang, H.(2023). Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis. eLife. DOI: 10.7554/eLife.83069
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