Immune Cell Phenotyping Using Flow Cytometry

A. Graham Pockley1, Gemma A. Foulds2, Julie A. Oughton3, Nancy I. Kerkvliet3, Gabriele Multhoff4

1 Electric Works, Sheffield, 2 Nottingham Trent University, Nottingham, 3 Oregon State University, Corvallis, 4 German Research Center for Environmental Health, Neuherberg
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.8
DOI:  10.1002/0471140856.tx1808s66
Online Posting Date:  November, 2015
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Fluorescent immunophenotyping uses fluorescently‐conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single‐cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single‐cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser‐based technology which analyzes the physical and fluorescent properties of cells in suspension in real‐time as they flow through the instrument. This approach has a number of advantages over other techniques that can be used for characterizing cell populations in single‐cell suspensions, in that it can nonsubjectively interrogate up to millions of cells and acquire data on the presence of different cell subpopulations and phenotypical changes within these populations in seconds. This unit describes basic procedures for the direct and indirect immunofluorescent staining of surface and intracellular proteins that are expressed by lymphoid cells which have been isolated from tissues or blood. Protocols for the resolution of dead cells and for the fixation of cells are also included. This unit also provides essential information relating to the selection and titration of antibodies, fluorochrome choice, spectral overlap and compensation, the use of controls, and the standardization of data acquisition and analysis. It also highlights new technologies and platforms that can be used to interrogate the presence of cell subpopulations and their phenotype to an even greater depth. © 2015 by John Wiley & Sons, Inc.

Keywords: flow cytometry; immune phenotyping; toxicology; cell analysis; methodology

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Table of Contents

  • Introduction
  • Basic Protocol 1: Direct Staining of Cells Prepared From Lymphoid Tissues
  • Alternate Protocol 1: Indirect Staining of Cells Prepared From Lymphoid Cells
  • Support Protocol 1: Preparation of Cells From Lymphoid Organs
  • Support Protocol 2: Cell Fixation
  • Basic Protocol 2: Staining of Peripheral Blood Leukocytes
  • Support Protocol 3: Assessment of Cell Viability
  • Support Protocol 4: Titration to Determine Optimum Antibody Concentration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Direct Staining of Cells Prepared From Lymphoid Tissues

  • Tissue of interest (e.g., lymphoid cells; see protocol 3)
  • Fc receptor (FcR)‐blocking immunoglobulin (Ig; e.g., normal rat IgG) solution, 200 μg/ml or specific Fc blocking reagents that are suitable for use in the species of interest
  • MAbs or isotype Igs directly conjugated to fluorochromes
  • PBS supplemented with 0.1% (w/v) sodium azide and bovine serum albumin (PAB; see recipe)
  • Micropipettor or multichannel pipettor with tips
  • 96‐well V‐bottom microtiter plates, disposable 12 × 75–mm polystyrene tubes, or microcentrifuge tubes
  • Centrifuge, refrigerated and equipped with a microtiter plate carrier
  • Vacuum aspiration system (optional)
  • Vortex equipped with a platform head for a microtiter plate
  • Titertube (Bio‐Rad Laboratories; optional)
  • Additional reagents and equipment for preparation of lymphocyte suspension to be analyzed (see protocol 3)

Alternate Protocol 1: Indirect Staining of Cells Prepared From Lymphoid Cells

  Additional Materials (see also protocol 1)
  • Unconjugated primary MAb or biotin‐labeled primary MAb
  • Fluorochrome‐conjugated anti‐Ig F(ab′) 2 fragment (for use with unconjugated primary MAb) or fluorochrome‐conjugated streptavidin (for use with biotin‐labeled primary MAb)

Support Protocol 1: Preparation of Cells From Lymphoid Organs

  • Lymphoid organ of interest
  • HBSS supplemented with 5% (v/v) FBS (5% HBSS; see recipe)
  • Endotoxin‐screened distilled water for cell culture (e.g., Gibco)
  • 10× HBSS (e.g., Sigma)
  • Ammonium chloride (ACK) lysing buffer (optional; see recipe)
  • 60 × 15–mm untreated culture dish
  • 25 × 75 × 1–mm frosted glass microscope slides
  • 15‐ml conical centrifuge tubes
  • 12 × 75–mm disposable cell culture tubes
  • Vortex
  • Cell counting platform (e.g., Coulter counter or hemacytometer)

Support Protocol 2: Cell Fixation

  Additional Materials (see also protocol 1)
  • 1% (w/v) formaldehyde: 10% (v/v) Ultrapure EM‐grade formaldehyde (e.g., Polysciences) diluted 1:10 in PAB (see recipe), or other commercially‐available fixation solutions
  • 40‐μm nylon mesh (optional)

Basic Protocol 2: Staining of Peripheral Blood Leukocytes

  • Animals of interest
  • Sodium heparin, 250 IU per ml in PBS ( appendix 2A)
  • PBS supplemented with 0.1% (w/v) sodium azide and bovine serum albumin (PAB; see recipe)
  • FcR‐blocking Ig solution, 2 mg/ml (note that concentration differs from that used in protocol 1)
  • Appropriate primary MAbs and (if necessary) secondary antibodies
  • Red blood cell/erythrocyte lysing solution, diluted appropriately according to the manufacturer's instructions.
  • 1‐ml syringe equipped with 22‐G needle
  • Cell counting platform (e.g., Coulter counter or hemacytometer)
  • Disposable 12 × 75–mm polystyrene tubes
  • Centrifuge
  • Vortex
NOTE: Staining conditions are similar to those noted in protocol 1. However, all staining procedures should be performed in 12 × 75–mm polystyrene tubes, not in microtiter plates.NOTE: Be sure to protect samples from light during incubation with fluorochrome‐conjugated MAbs.

Support Protocol 3: Assessment of Cell Viability

  • Cells of interest
  • Nuclear staining compound dissolved in PBS ( appendix 2A): 200 μg/ml PI, 250 μg/ml 7‐AAD, 250 μg/ml TO‐PRO‐3 (e.g., Molecular Probes), or 200 μg/ml PY(G)
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer

Support Protocol 4: Titration to Determine Optimum Antibody Concentration

  • Lymphoid cell suspension of interest (see protocol 3)
  • Fc receptor (FcR)‐blocking immunoglobulin (Ig) solution, 200 μg/ml
  • Fluorochrome‐conjugated antibody solutions to be tested
  • 96‐well microtiter plate
  • Centrifuge fitted with a microtiter plate adapter
  • Vortex fitted with a microtiter plate adapter
  • Flow cytometer with appropriate analysis software
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