High‐Throughput Particle Uptake Analysis by Imaging Flow Cytometry

Asya Smirnov1, Michael D. Solga2, Joanne Lannigan1, Alison K. Criss1

1 Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia, 2 University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.22
DOI:  10.1002/cpcy.19
Online Posting Date:  April, 2017
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Abstract

Quantifying the efficiency of particle uptake by host cells is important in the fields of infectious diseases, autoimmunity, cancer, developmental biology, and drug delivery. Here we present a protocol for high‐throughput analysis of particle uptake by imaging flow cytometry, using the bacterium Neisseria gonorrhoeae attached to and internalized by neutrophils as an example. Cells are exposed to fluorescently labeled bacteria, fixed, and stained with a bacteria‐specific antibody of a different fluorophore. Thus, in the absence of a permeabilizing agent, extracellular bacteria are double‐labeled with two fluorophores while intracellular bacteria remain single‐labeled. A spot count algorithm is used to determine the number of single‐ and double‐labeled bacteria in individual cells, to calculate the percent of cells associated with bacteria, percent of cells with internalized bacteria, and percent of cell‐associated bacteria that are internalized. These analyses quantify bacterial association and internalization across thousands of cells and can be applied to diverse experimental systems. © 2017 by John Wiley & Sons, Inc.

Keywords: attachment; bacteria; internalization; imaging flow cytometry; phagocytosis

     
 
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Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: Differential Staining of Cell‐Bound and Internalized Bacteria Associated with Adherent Cells
  • Alternate Protocol 1: Differential Staining of Cell‐Bound and Internalized Bacteria from Cells in Suspension
  • Alternate Protocol 2: Differential Staining of Cell‐Bound and Internalized Bacteria in Cells at Low Temperature
  • Alternate Protocol 3: Differential Staining of Cell‐Bound and Internalized Bacteria after Blocking Actin Polymerization
  • Alternate Protocol 4: Differential Staining of Cell‐Bound and Internalized Bacteria in Fixed Cells
  • Support Protocol 1: Labeling Bacteria with Carboxylfluorescein Succinimidyl Ester (CFSE)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Differential Staining of Cell‐Bound and Internalized Bacteria Associated with Adherent Cells

  Materials
  • Cultured adherent target cells (e.g., primary human neutrophils) in 6‐well tissue culture plates
  • CFSE‐labeled N. gonorrhoeae in suspension (see protocol 6Support Protocol)
  • Cell culture medium: e.g., RPMI 1640 with 10% (v/v) fetal bovine serum
  • 4% (w/v) paraformaldehyde solution (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • 10% (v/v) normal goat serum (see recipe)
  • Bacteria‐specific antibody conjugated to the DyLight 650 (Thermo Fisher Scientific; used according to the manufacturer's protocol)
  • CO 2 tissue culture incubator
  • Refrigerated clinical centrifuge with swing‐bucket rotor, microplate carriers, and adapters for 15‐ml conical tubes
  • Cell scrapers
  • 15‐ml conical tubes
  • Amnis ImageStreamX system with the INSPIRE acquisition and IDEAS data analysis software (EMD Millipore)

Alternate Protocol 1: Differential Staining of Cell‐Bound and Internalized Bacteria from Cells in Suspension

  Materials (also see Basic Protocol)
  • Cells cultured in suspension in appropriate cell growth medium

Alternate Protocol 2: Differential Staining of Cell‐Bound and Internalized Bacteria in Cells at Low Temperature

  Materials (also see Basic Protocol)
  • 10 mg/ml cytochalasin D (see recipe)
  • DMSO

Alternate Protocol 3: Differential Staining of Cell‐Bound and Internalized Bacteria after Blocking Actin Polymerization

  Materials (also see Basic Protocol)
  • 2% (w/v) paraformaldehyde in PBS, pH 7.4 (see recipe)

Alternate Protocol 4: Differential Staining of Cell‐Bound and Internalized Bacteria in Fixed Cells

  Materials
  • Bacteria grown to log phase in the appropriate liquid culture media
  • 10 mg/ml CFDA‐SE (see recipe)
  • PBS containing 5 mM MgSO 4 (see recipe)
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Figures

Videos

Literature Cited

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