Preparation of Cells and Reagents for Flow Cytometry

Kevin Holmes1, Larry M. Lantz1, B.J. Fowlkes1, Ingrid Schmid2, Janis V. Giorgi2

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 2 UCLA School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.3
DOI:  10.1002/0471142735.im0503s44
Online Posting Date:  November, 2001
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Abstract

Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent‐labeled antibodies or ligands that bind to specific cell‐associated molecules. A procedure for direct and indirect staining of single‐cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single‐cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.

     
 
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Table of Contents

  • Basic Protocol 1: Immunofluorescence Staining of Single‐Cell Suspensions for Detection of Surface Antigens
  • Basic Protocol 2: Immunofluorescence Staining of Fixed and Permeabilized Single‐Cell Suspensions for Detection of Intracellular Antigens
  • Support Protocol 1: Labeling Antibody with Fluorescein Isothiocyanate (FITC)
  • Support Protocol 2: Labeling Antibody Using Long‐Armed Biotin
  • Support Protocol 3: Labeling Antibody with Texas Red
  • Support Protocol 4: Labeling Antibody with Phycobiliproteins
  • Alternate Protocol 1: Immunofluorescence Staining of Unfixed Cells for Detection of Intracellular Antigens
  • Alternate Protocol 2: Staining of Nonviable Cells with 7‐Aminoactinomycin D for Dead‐Cell Discrimination
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Immunofluorescence Staining of Single‐Cell Suspensions for Detection of Surface Antigens

  Materials
  • Sample material: lymphoid tissue (unit 3.1) or single‐cell suspension of human peripheral blood (unit 7.1)
  • recipeStaining buffer (see recipe), 4°C
  • Labeled or unlabeled antibody (see Support Protocols protocol 31 to protocol 64) diluted to the appropriate concentration as determined by titering (see )
  • recipePropidium iodide (PI) solution (optional; see recipe)
  • recipeFixation solution (optional; prepare immediately before use; see recipe)
  • 12 × 15–mm round‐bottom test tubes or 96‐well round‐bottom microtiter plates
  • 100‐µm nylon mesh (optional)
  • Sorvall RT‐6000B with H‐1000B rotor (or equivalent)
  • Additional reagents and equipment for trypan blue exclusion ( appendix 3B)

Basic Protocol 2: Immunofluorescence Staining of Fixed and Permeabilized Single‐Cell Suspensions for Detection of Intracellular Antigens

  Materials
  • Cell sample: mononuclear cells derived from human or murine peripheral blood, bone marrow, thymus or spleen; cells grown in suspension cultures; or dissociated tissues (unit 3.1)
  • PBS ( appendix 2A) without Ca2+ and Mg2+, 4°C
  • recipeFixation solution (see recipe), 4°C
  • recipePermeabilization solution (see recipe)
  • Fluorochrome‐labeled, biotin‐labeled, or unlabeled antibody (see protocol 3Support Protocols 1 to protocol 64 or use commercial supplier) appropriately diluted in recipestaining buffer (see recipe)
  • recipeWashing buffer (see recipe)
  • Second‐step fluorochrome‐labeled antibody or avidin/streptavidin
  • PBS containing 1 mg/ml propidium iodide (PI) or 7‐aminoactinomycin D (7‐AAD; optional; see recipes for recipePI and recipe7‐AAD stocks)
  • 12 × 15–mm round‐bottom test tubes
  • Sorvall H‐1000B rotor or equivalent
  • 62‐µm nylon mesh (Small Parts; optional)

Support Protocol 1: Labeling Antibody with Fluorescein Isothiocyanate (FITC)

  Materials
  • 1 to 2 mg/ml purified monoclonal antibody (units 2.4 & 2.5)
  • recipeFITC labeling buffer, 4°C (see recipe; prepare ≤2 weeks before use)
  • 5 mg/ml FITC in anhydrous dimethylsulfoxide (DMSO)
  • recipeDialysis buffer, 4°C (see recipe)
  • Sephadex G‐25 column (Pharmacia Biotech PD‐10; optional)
  • Additional reagents and equipment for dialysis ( appendix 3H)

Support Protocol 2: Labeling Antibody Using Long‐Armed Biotin

  • recipeSuccinimide ester labeling buffer (see recipe)
  • 10 mg/ml long‐armed biotin (Zymed) in anhydrous dimethylsulfoxide (DMSO)

Support Protocol 3: Labeling Antibody with Texas Red

  Materials
  • 1 to 2 mg/ml purified monoclonal antibody
  • recipeSuccinimide ester labeling buffer, 4°C (see recipe)
  • 5 mg/ml Texas Red–X succinimidyl ester (Molecular Probes) in N,N‐dimethylformamide (DMF)
  • recipeDialysis buffer, 4°C (see recipe)
  • recipeStabilizing buffer (see recipe)
  • Dialysis tubing
  • Sephadex G‐25 column (Pharmacia Biotech; optional)

Support Protocol 4: Labeling Antibody with Phycobiliproteins

  • 10 to 25 mg/ml phycoerythrin (PE)
  • recipeCoupling buffer, pH 5.5 and 7.5 (see recipe)
  • Sulfhydryl addition reagent: N‐succinimidyl‐S ‐acetylthioacetate (SATA; Calbiochem): store under nitrogen after opening
  • Dimethylformamide (DMF)
  • Heterobifunctional cross‐linker: γ‐maleimidobutyric acid N‐hydroxysuccinimide ester (GMBS, Calbiochem; store under nitrogen after opening)
  • recipeDeacetylation buffer (see recipe)
  • Tetrahydrofuran (THF)
  • Cysteine
  • recipeRunning buffer, degassed (see recipe)
  • AcA 34 column (IBF Biotechnics)

Alternate Protocol 1: Immunofluorescence Staining of Unfixed Cells for Detection of Intracellular Antigens

  • 0.3% and 0.1% (w/v) saponin (Sigma) in PBS ( appendix 2A): store ≤1 month in amber container at 4°C
  • 0.3% saponin in PBS with 10 µg/ml PI or 20 µg/ml 7‐AAD (optional): add recipePI or recipe7‐AAD stock to appropriate concentration (see reciperecipes); prepare fresh before use and protect solution from light

Alternate Protocol 2: Staining of Nonviable Cells with 7‐Aminoactinomycin D for Dead‐Cell Discrimination

  • recipe1 mg/ml 7‐aminoactinomycin D (7‐AAD) stock solution (see recipe)
  • Fixation solution containing 80 µg/ml recipeactinomycin D (AD; see recipe), 4°C
  • 0.2% and 0.1% (v/v) Tween 20 in PBS containing 10 µg/ml AD
  • recipeStaining buffer (see recipe) containing 10 µg/ml AD
  • Fluorochrome‐labeled antibody in 0.3% (w/v) saponin/PBS containing 10 µg/ml AD
  • 0.1% saponin in PBS containing 10 µg/ml AD
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Figures

Videos

Literature Cited

Literature Cited
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Key References
   Clevenger and Shankey, 1993. See above.
  In‐depth discussion of background information and excellent review of intracellular staining methods for flow cytometry and their application.
   Jacob et al., 1991. See above.
   Describes and shows examples of its application.
   Schmid et al., 1991. See above.
  Describes and provides examples of its application.
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