Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Summary

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Abstract

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Introduction

Apoptosis, or programmed cell death, is a highly-regulated physiological process that occurs in most multicellular organisms and is crucial for their development and homeostasis¹. In addition to being involved in normal cell turnover and embryonic development, apoptosis enables the elimination of infected or damaged cells from tissues and can be triggered in response to infection, inflammation, cancer, and also by medical interventions such as radiotherapy or steroids¹. Apoptotic cells expose "eat-me" signals on their cell surface which are recognized by receptors on a range of professional and non-professional p....

Protocol

Collection of blood from healthy volunteers was approved by the Health Science Research Ethics Board of the University of Western Ontario. Venipuncture was performed in accordance with the guidelines of the Tri-Council Policy Statement on human research.

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 immedi....

Results

Overnight culture of Jurkat cells with 1 µM staurosporine results in apoptosis of >95% of cells, which can be confirmed with Annexin V staining (Figure 1). Other cell types can be used for these experiments, although the concentration of staurosporine and the duration of staurosporine treatment will need to be optimized for each cell line. For reliable detection and quantification of efferocytosis, >80% of cells should be apoptotic prior to adding them .......

Discussion

The methods outlined in this protocol enable the imaging and quantification of the dynamic efferocytic process, using both fixed-cell and live-cell approaches. These approaches offer several advantages over commonly employed flow cytometry-based methods^23,24. The use of inside-out staining with fixed samples provides a more robust and accurate quantification of the rate and extent of efferocytosis — indeed, many flow cytometry-based methods simply label apo.......

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