Basic Multicolor Flow Cytometry

Zofia Maciorowski1, Pratip K. Chattopadhyay2, Paresh Jain3

1 Institut Curie, Paris, 2 Laura and Isaac Perlmutter Cancer Center, NYU‐Langone Medical Center, New York, New York, 3 BDB Asia‐Pacific, BD Life Sciences, Gurgaon
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.4
DOI:  10.1002/cpim.26
Online Posting Date:  April, 2017
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Abstract

Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell sorting for further experimentation. This unit covers the tools needed by the beginning immunologist to plan and run multicolor experiments, with information on fluorochromes and their characteristics, spectral spillover, compensation and spread, instrument and reagent variables, and the basic elements of multicolor panel design. Protocols to quantify and maximize sensitivity by titration of reagents and optimization of instrument settings, as well as basic surface and intracellular cell staining, are included. © 2017 by John Wiley & Sons, Inc.

Keywords: flow cytometry; multicolor; fluorescent

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Stain Index
  • Basic Protocol 2: Determination of Best Gain or PMT Voltage Settings (Voltration)
  • Basic Protocol 3: Antibody Titration
  • Basic Protocol 4: Compensation
  • Basic Protocol 5: Surface Staining
  • Basic Protocol 6: Surface and Intracellular Staining
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Stain Index

  Materials
  • Compensation beads appropriate to your antibody; some compensation beads are generic and will bind any species of antibody, others are species‐specific and will bind mouse, rat, or hamster antibodies
  • Staining buffer: phosphate‐buffered saline (PBS; Gibco, cat. no. 14190‐094) without Ca or Mg, containing 1% bovine serum albumin (BSA; Sigma‐Aldrich)
  • Fluorochrome‐labeled antibody
  • 12 × 15 mm round‐bottom tubes
  • Centrifuge
  • Flow cytometer (see Chapter 5 in this manual)
  • Additional reagents and equipment for flow cytometry (see Chapter 5 in this manual)

Basic Protocol 2: Determination of Best Gain or PMT Voltage Settings (Voltration)

  Materials
  • Antibody of interest labeled with fluorochrome: at a concentration 4× the manufacturer's recommendation, can be higher or lower if desired
  • Staining buffer: phosphate‐buffered saline (PBS; Gibco, cat. no. 14190‐094) without Ca or Mg, containing 1% bovine serum albumin (BSA; Sigma Aldrich)
  • Sample: cell suspension (1–5 × 106 cells/ml); the cells should be prepared, unfixed or fixed, in the same way as they will for the multicolor experiment
  • 12 × 15–mm round‐bottom tubes or 96‐well u‐bottom plates
  • Centrifuge
  • Flow cytometer (see Chapter 5 in this manual)
  • Additional reagents and equipment for data acqusition by flow cytometry and data analysis ( protocol 1)

Basic Protocol 3: Antibody Titration

  Materials
  • Staining buffer: phosphate‐buffered saline (PBS; Gibco, cat. no. 14190‐094) without Ca or Mg, containing 1% bovine serum albumin (BSA; Sigma‐Aldrich)
  • Sample: single‐cell suspension (1–5 × 106 cells/ml in staining buffer; use more cells if looking for rare populations)
  • Fluorochrome‐labeled antibodies, titrated as described in protocol 3
  • Compensation beads appropriate to your antibody; some compensation beads are generic and will bind any species of antibody, others are species‐specific and will bind mouse, rat, or hamster antibodies
  • Live/dead cell marker (PI, DAPI, 7‐AAD) appropriate to your fluorochrome panel, usually ∼2 to 10 μg/ml final concentration
  • 12 × 15–mm round bottom tubes
  • Centrifuge
  • Flow cytometer (also see Chapter 5 in this manual)

Basic Protocol 4: Compensation

  Materials
  • Staining buffer (optimized for your system: see annotation to step 9): phosphate‐buffered saline (PBS; Gibco, cat. no. 14190‐094) without Ca or Mg, containing 1% bovine serum albumin (BSA; Sigma‐Aldrich)
  • Sample: single‐cell suspension (1–5 × 106 cells/ml in staining buffer; use more cells if looking for rare populations)
  • Fluorochrome‐labeled antibodies, titrated as described in protocol 3
  • Live/dead cell fixable marker appropriate to your fluorochrome panel
  • Compensation beads appropriate to your antibody; some compensation beads are generic and will bind any species of antibody, others are species‐specific and will bind mouse, rat, or hamster antibodies
  • Fixation and permeabilization buffers optimized for your system
  • 12 × 15 –mm round‐bottom tubes
  • Centrifuge
  • Flow cytometer (also see Chapter 5 in this manual)
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Figures

Videos

Literature Cited

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Spectral viewers
  http://www.biolegend.com/spectraanalyzer
  BioLegend Spectral Viewer.
  https://www.bdbiosciences.com/br/research/multicolor/spectrum_viewer/index.jsp
  BD Biosciences Spectral Viewer.
  https://www.thermofisher.com/fr/fr/home/life‐science/cell‐analysis/labeling‐chemistry/fluorescence‐spectraviewer.html#
  Thermo Fisher spectral viewer.
Panel design programs
  https://www.fluorish.com/
  Fluorish panel design program.
  https://fluorofinder.com/
  Fluorofinder panel design program.
  https://www.bdbiosciences.com/sg/paneldesigner/index.jsp
  BD Biosciences panel design program.
Fluorochrome brightness
  http://www.biolegend.com/brightness_index
  Biolegend brightness index.
  http://static.bdbiosciences.com/documents/multicolor_fluorochrome_laser_chart.pdf?_ga=1.193693357.1447862526.1480066966
  BD Biosciences fluorochrome relative brightness.
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