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

Published: April 30, 2023

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 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 …

Divulgazioni

The authors have nothing to disclose.

Materials

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

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Citazione di questo articolo
Fixed Cell Efferocytosis Assay: A Method to Study Efferocytic Uptake of Apoptotic Cells by Macrophages Using Fluorescence Microscopy. J. Vis. Exp. (Pending Publication), e21236, doi: (2023).

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