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 …
Divulgaciones
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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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).