Measurement of Phagocytic Engulfment of Apoptotic Cells by Macrophages Using pHrodo Succinimidyl Ester

Monowar Aziz1, Weng‐Lang Yang1, Ping Wang1

1 Center for Immunology and Inflammation, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‐LIJ School of Medicine, Manhasset, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.31
DOI:  10.1002/0471142735.im1431s100
Online Posting Date:  February, 2013
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Abstract

Considerable interest has emerged towards phagocytosis of apoptotic cells, due to its intricate molecular mechanisms and important regulatory functions in development, homoeostasis, and immune tolerance. Impaired clearance of apoptotic cells leads to immune‐mediated disorders. Current quantification methods of the engulfment of apoptotic cells by macrophages are potentially flawed by several limitations. Adherent macrophage populations are overlaid with apoptotic targets in suspension and then co‐cultured for a definite period, which may give rise to two different features: (1) engulfed and (2) non‐engulfed macrophages that are surface‐bound cell populations. Rigorous washing to dislodge surface‐bound apoptotic cells before assessment of phagocytosis may lead to loss of phagocytes, thereby skewing the apparent magnitude of the overall phagocytic response. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. In this unit, we demonstrate the use of pHrodo‐succinimidyl ester (SE), a pH‐sensitive fluorescent dye, to label the apoptotic cells for monitoring the phagocytosis. After engulfment, the intensity of pHrodo light emission will be elevated due to the pH change inside of macrophages. The shift of pHrodo light emission can be detected by a flow cytometer or using a fluorescence microscope. Curr. Protoc. Immunol. 100:14.31.1‐14.31.8. © 2013 by John Wiley & Sons, Inc.

Keywords: apoptotic cells; macrophage; pHrodo‐SE; phagocytosis; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Phagocytic Engulfment of Apoptotic Cells by pHrodo‐SE Using Flow Cytometry
  • Alternate Protocol 1: Detection of Phagocytic Engulfment of Apoptotic Cells by pHrodo‐SE Using Fluorescence Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of Phagocytic Engulfment of Apoptotic Cells by pHrodo‐SE Using Flow Cytometry

  Materials
  • Animals: BALB/c or C57BL/6 mice, specific‐pathogen‐free, typically 6 to 8 weeks old (Taconic)
  • Phosphate‐buffered saline (PBS; pH 7.2‐7.4; Invitrogen, cat. no. 20012‐027)
  • Red blood cells (RBC) lysis solution (see recipe)
  • Dulbecco's modified Eagle medium (DMEM; Invitrogen, cat. no. 11965‐092)
  • Fetal bovine serum (FBS; MP Biomedicals, cat. no. 092916754)
  • Penicillin/streptomycin (100×; Invitrogen, cat. no. 15140‐122)
  • 200 mM L‐glutamine (Invitrogen, cat. no. 25030‐081)
  • Dexamethasone (see recipe)
  • Annexin V‐FITC Apoptosis Detection kit (BD Biosciences, cat. no. 556570) containing:
    • Annexin V
    • Propidium iodide
    • Binding buffer
  • pHrodo‐SE (see recipe)
  • OPTI‐MEM (Invitrogen, cat. no. 31985‐062)
  • 1% paraformaldehyde (PFA; see recipe)
  • Frosted glass slides
  • Nylon mesh (70 µm; BD Falcon, cat. no. 352350)
  • 50‐ml centrifuge tubes
  • Centrifuge
  • 37°C, 5% CO 2 humidified incubator
  • 60‐mm tissue culture dishes (BD Falcon, cat. no. 354102)
  • Cell lifter
  • FACS tubes
  • Additional reagents and equipment for anesthesia (unit 1.4), removal of lymphoid organs (unit 1.9), determining cell number using trypan blue dye exclusion ( appendix 3A), isolation of mouse thioglycollate‐induced peritoneal macrophages (unit 14.1), and flow cytometric analysis of cells (units 5.1& 5.4)

Alternate Protocol 1: Detection of Phagocytic Engulfment of Apoptotic Cells by pHrodo‐SE Using Fluorescence Microscopy

  • Macrophages (see unit 14.1)
  • 4 ×104 pHrodo‐SE‐labeled apoptotic thymocytes (see protocol 1)
  • FITC‐anti‐mouse CD11b (cat. no. 553310; Integrin‐α M chain Mac‐1; BD Biosciences)
  • 2‐well culture slides (BD Falcon, cat. no. 354114)
  • Fluorescence microscope (Nikon Eclipse Ti‐S)
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Figures

  •   FigureFigure 14.31.1 Detection of phagocytosis of pHrodo‐SE‐labeled apoptotic cells by flow cytometry. Macrophages (106 cells) were co‐cultured with pHrodo‐SE‐labeled apoptotic cells (4×106 cells) for 2 hr. After PBS wash, the adherent cells were collected for assessment of pHrodo‐SE using flow cytometry. (A) pHrodo‐SE‐labeled apoptotic cells were gated according to their forward and side scatter properties and then used to make a cut‐off point of fluorescence emission. The histogram represents the median fluorescence intensity (MFI). (B) After co‐culture, macrophage cells were gated and then the fluorescence intensities were measured and expressed as MFI.
  •   FigureFigure 14.31.2 Fluorescence microscopic detection of phagocytosis of pHrodo‐SE‐labeled apoptotic cells. After co‐culturing the macrophages (104 cells) with pHrodo‐SE‐labeled apoptotic cells (4×104 cells) for 2 hr, immunofluorescence studies were performed by staining the macrophages with the FITC‐anti‐mouse‐CD11b (green). (A) Apoptotic cells that are not engulfed by the FITC‐stained macrophages showing dim fluorescence. (B) Apoptotic cells that are truly engulfed by the FITC‐positive macrophages showing intense orange fluorescence. Original magnification, ×400.
  •   FigureFigure 14.31.3 Schematic chart of processing the assay for detecting the engulfment of apoptotic cells by macrophages.

Videos

Literature Cited

Literature Cited
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