Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

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

1. 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.
2. Obtain single-cell suspension by grinding the whole thymus against two frosted ends of the microscope slides and then filter the suspension through a 100 ....

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 f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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