Efferocytosis assay

 Apoptotic thymocytes (ACs) acquisition

1. C57BL/6 wild-type mice were euthanized by cervical dislocation, followed by disinfection through immersion in 75% alcohol.
2. The thorax was incised along the midclavicular line bilaterally, exposing the white thymic tissue located posterior to the sternum.
3. 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. 
4. Red blood cells were eliminated using a red blood cell lysing solution.
5. After PBS washing, the cells were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).
6. 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

1. 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.
2. CFSE-labeled apoptotic thymocytes were added at a 10:1 ratio to the macrophage cultures and incubated for an additional 120 minutes.
3. Cells were thoroughly washed three times with PBS to remove non-engulfed thymocytes.
4. 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.
5. 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.
6. Following immunofluorescence staining and mounting with DAPI, samples were visualized using a fluorescence microscope.
7. 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%).