Fixed Cell Efferocytosis Assay: A Method to Study Efferocytic Uptake of Apoptotic Cells by Macrophages Using Fluorescence Microscopy

Abstract

Source: Taruc, K., et al. Quantification of Efferocytosis by Single-cell Fluorescence Microscopy. J. Vis. Exp. (2018).

In this video, we demonstrate the efferocytosis assay to study the clearance of apoptotic cells using confocal microscopy. Efferocytosis is a multi-step process where apoptotic cells are engulfed by phagocytes and degraded within vesicles called efferosomes. This protocol provides a method to visualize internalized and non-internalized portions of individual apoptotic cells and quantify efferocytosis.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Culture and Preparation of the THP-1 Monocyte Cell Line

1. Culture THP-1 monocytes as a suspension culture in T25 flasks at 37 °C + 5% CO₂. Cells should be grown in 5 mL of Roswell Park Memorial Institute 1640 (RPMI 1640) + 10% Fetal Bovine Serum (FBS).
2. Each day, suspend cells evenly throughout the growth media by gently shaking the flask, then immediately count cells with a hemocytom....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640 Media	Wisent	3500-000-EL
DMEM Media	Wisent	319-005-CL
Fetal Bovine Serum (FBS)	Wisent	080-150
PBS	Wisent	311-010-CL
18 mm circular glass coverslips #1.5 thickness	Electron Microscopy Sciences	72290-08	Size and shape of coverslip is not critical, but 18 mm fit into the wells of a standard 12-well plate which simplifies cell culture
Staurosporine	Cayman Chemical	81590	Dissolve in DMSO at 1 mM (1,000x stock solution)
Annexin V-Alexa 488	ThermoFisher	R37174
EZ-Link NHS-Biotin	ThermoFisher	20217	Store in a dessicator. Do not prepare a stock solution.
DMSO	Sigma-Aldrich	D2650
CellTrace FarRed	ThermoFisher	C34572
CellTrace Orange	ThermoFisher	C34851
Hoescht 33342	ThermoFisher	62249
FITC-Streptavadin	ThermoFisher	SA1001
Lympholyte-poly cell sepration medium	Cedarlane Labs	CL5071
Recombinant Human M-CSF	Peprotech	200-04
Recombinant Human IL-4	Peprotech	300-25
J774.2 Macrophage Cell Line	Sigma-Aldrich	85011428-1VL
THP-1 Human Monocyte Cell Line	ATCC	TIB-202
Jurkat T Cell Line	ATCC	TIB-152