Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Published: November 30, 2023

Abstract

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.

Protocol

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 Euthanize two naïve C57/B6 mice by CO2 inhalation for 10 min and dissect to open the chest cavity, remove (pull out) the thymus with curved fine-tip forceps into tissue culture petri dish containing 10 mL of RPMI1640 medium. Obta…

Representative Results

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 percen…

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages. J. Vis. Exp. (Pending Publication), e21786, doi: (2023).

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