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Immunology and Infection

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Published: August 18th, 2018

DOI:

10.3791/58149

1Department of Microbiology and Immunology and the Center for Human Immunology, University of Western Ontario, 2Institute of Infection and Global Health, University of Liverpool, 3Department of Respiratory Research, Aintree University Hospital NHS Foundation Trust

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.

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.

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....

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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.......

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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 addin.......

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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 methods23,24. 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.......

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This study was funded by Canadian Institutes of Health Research (CIHR) Operating Grant MOP-123419, Natural Sciences and Engineering Research Council of Canada Discovery Grant 418194, and an Ontario Ministry of Research and Innovation Early Research Award to BH. DGW contributed some of the images presented, to the optimization of the protocols and to the writing of the manuscript; he was funded by a pump-priming grant from the university of Liverpool. CY is funded by a Vanier Graduate Scholarship and CIHR MD/PhD Studentship. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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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

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