Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Kyle Taruc; Charles Yin; Daniel G. Wootton; Bryan Heit

doi:10.3791/58149

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

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06:15 min

August 18, 2018

DOI: 10.3791/58149-v

Kyle Taruc¹, Charles Yin¹, Daniel G. Wootton^2,3, Bryan Heit¹

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

Overview

This article presents a fluorescence microscopy protocol for quantifying efferocytosis, the process of phagocytic removal of apoptotic cells. The method allows for the assessment of efferocytic signaling pathways and provides clear quantification of apoptotic cell uptake.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Immunology

Background

Efferocytosis is crucial for maintaining cellular homeostasis.
It involves the recognition and internalization of apoptotic cells by phagocytes.
Understanding the mechanisms of efferocytosis can provide insights into various diseases.
This method can be applied to different cell types, including immune and cancer cells.

Purpose of Study

To quantify the uptake of apoptotic cells by immune cells.
To investigate the role of signaling molecules in efferocytosis.
To provide a reliable method for studying efferocytosis in various cell types.

Methods Used

Preparation of human macrophages and apoptotic Jurkat cells.
Counting apoptotic cells using a hemocytometer.
Fluorescence microscopy for visualization and quantification.
Optimization of microscope acquisition settings for accurate results.

Main Results

The method allows for clear quantification of apoptotic cell uptake.
Partial or piecemeal uptake of apoptotic cells can be assessed.
Insights into efferocytosis mechanisms can be gained through this approach.
The technique is applicable to various cell types beyond immune cells.

Conclusions

This fluorescence microscopy protocol enhances the understanding of efferocytosis.
It provides a valuable tool for researchers studying cell death and clearance.
Future applications may extend to other fields of research involving cell interactions.

Frequently Asked Questions

What is efferocytosis?

Efferocytosis is the process by which phagocytic cells remove apoptotic cells to maintain homeostasis.

Why is quantifying efferocytosis important?

Quantifying efferocytosis helps understand the mechanisms of cell clearance and its implications in various diseases.

What types of cells can this method be applied to?

This method can be applied to immune cells, epithelial cells, and cancer cells.

What are the challenges in using this method?

Optimizing microscope acquisition settings can be difficult for individuals new to this method.

What advantage does this method have over others?

It provides clear quantification of apoptotic cell uptake, including partial uptake, which may not be visible with other methods.

How are apoptotic cells prepared for this method?

Apoptotic cells are prepared by using human macrophages and Jurkat cells, followed by counting with a hemocytometer.

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.

This method can be used to answer key questions in the efferocytosis field such as the role of signaling molecules in mediating the uptake of apoptotic cells. The main advantage of this technique is that it provides a clear quantification of apoptotic cell uptake including partial or piecemeal apoptotic cell uptake which isn't always apparent with other methods. Though this method can provide insight into efferocytosis by immune cells it can also be applied to other cell types such as epithelial or cancer cells.

Generally, individuals new to this method will struggle because of the difficulty in optimizing microscope acquisition settings. After preparing human macrophages and apoptotic Jurkat cells, use a hemocytometer to count the apoptotic cells. Then transfer a sufficient quantity of apoptotic cells to a 1.5 milliliter microcentrifuge tube.

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