Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of CFSE-labeled apoptotic thymocytes
2. Preparation of peritoneal macrophages
3. Co-culture of peritoneal macrophages with apoptotic thymocytes
Figure 1. Percentage of phagocytosis of apoptotic thymocytes by peritoneal macrophages. Apoptotic thymocytes were induced by incubating with 1 μM of staurosporine for 4 h and added into macrophage culture at a ratio as indicated in the figure panels: (A) 1:0; (B) 1:1; (C) 1:2; (D) 1:4; (E) 1:6; (F) 1:8; (G) 1:10; (H) 1:12. Data were acquired with a flow cytometer. The percentage of phagocytic macrophages was analyzed using the software associated with the flow cytometer.
The authors have nothing to disclose.
Ack lysing buffer | GIBCO | A10492 | |
Annexin V/7-AAD | BD Pharmingen | 559763 | |
Anti-Mer antibody | R&D Systems | BAF591 | |
CD11b-PE (clone M1/70) | BD Pharmingen | 553311 | |
CFSE | Invitrogen | C1157 | |
DMSO | Sigma-Aldrich | D-2650 | |
EDTA (0.5 mM) | GIBCO | 15575-020 | |
FACS tubes | BD Biosciences | 352017 | |
Horse Serum (Heat-inactivated) | Invitrogen | 26050088 | |
PBS, 1x | Corning | 21040CV | |
RPMI-1640 | Corning | 10040CV | |
RXDX-106 | Selleck Chemicals | CEP-40783 | |
Staurosprine (100mg) | Fisher Scientific | BP2541-100 | Add 214.3 ml of DMSO into 100mg to make 1mM stocking solution |