Cytometry > Flow Cytometry

User Ratings

Your rating: None
Your rating: None (1 vote)
Your rating: None
 Add your comments

Preparation of Cells and Reagents for Flow Cytometry

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

1National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
2UCLA 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
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • 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
  • Bibliography
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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)
  • Staining buffer (see recipe), 4°C
  • Labeled or unlabeled antibody (see Support Protocols 1 to 4) diluted to the appropriate concentration as determined by titering (see Critical Parameters)
  • Propidium iodide (PI) solution (optional; see recipe)
  • Fixation 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
  • Fixation solution (see recipe), 4°C
  • Permeabilization solution (see recipe)
  • Fluorochrome-labeled, biotin-labeled, or unlabeled antibody (see Support Protocols 1 to 4 or use commercial supplier) appropriately diluted in staining buffer (see recipe)
  • Washing 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 PI and 7-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)
  • FITC labeling buffer, 4°C (see recipe; prepare £2 weeks before use)
  • 5 mg/ml FITC in anhydrous dimethylsulfoxide (DMSO)
  • Dialysis 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

 Additional Materials (also see Support Protocol 1)
  • Succinimide 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
  • Succinimide ester labeling buffer, 4°C (see recipe)
  • 5 mg/ml Texas Red–X succinimidyl ester (Molecular Probes) in N,N-dimethylformamide (DMF)
  • Dialysis buffer, 4°C (see recipe)
  • Stabilizing buffer (see recipe)
  • Dialysis tubing
  • Sephadex G-25 column (Pharmacia Biotech; optional)

Support Protocol 4: Labeling Antibody with Phycobiliproteins

 Additional Materials (also see Support Protocol 1)
  • 10 to 25 mg/ml phycoerythrin (PE)
  • Coupling 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: -maleimidobutyric acid N-hydroxysuccinimide ester (GMBS, Calbiochem; store under nitrogen after opening)
  • Deacetylation buffer (see recipe)
  • Tetrahydrofuran (THF)
  • Cysteine
  • Running buffer, degassed (see recipe)
  • AcA 34 column (IBF Biotechnics)

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

 Additional Materials (also see Basic Protocol 2)
  • 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 PI or 7-AAD stock to appropriate concentration (see recipes); prepare fresh before use and protect solution from light

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

 Additional Materials (also see Basic Protocol 2)
  • 1 mg/ml 7-aminoactinomycin D (7-AAD) stock solution (see recipe)
  • Fixation solution containing 80 µg/ml actinomycin D (AD; see recipe), 4°C
  • 0.2% and 0.1% (v/v) Tween 20 in PBS containing 10 µg/ml AD
  • Staining 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
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 5.3.1
    (A) Forward scatter (FSC) versus side scatter (SSC) dot plot of untreated peripheral blood mononuclear cells (PBMC). For panels B-D, PBMC were first stained for CD3 with a phycoerythrin (PE)–conjugated antibody. They were then fixed and permeabilized as described in Basic Protocol Basic Protocol 2 and stained with either mouse IgG1-FITC control or anti-Bcl-2-FITC monoclonal antibody (Dako). (B) FSC versus SSC dot plot with the gate set around lymphocytes. (C) Single-parameter histogram of CD3-PE fluorescence of cells within the scatter gate with a gate set on CD3+ cells. (D) Overlay of mouse IgG1 fluorescence and Bcl-2 fluorescence of CD3+ cells within the scatter gate.

    View Image
  • Figure 5.3.2
    Human leukemic pre-T cells (SUP-T3) cell-surface stained with either mouse IgG1-FITC control or CD3-FITC monoclonal antibody (panels A-D). 7-AAD was added for dead cell discrimination, where indicated, at a concentration of 1 µg/ml. (A) FSC versus SSC dot plot. (B) FSC versus 7-AAD fluorescence dot plot with the gate set around live cells. (C) Mouse IgG1-FITC (background) fluorescence of cells within the gate. (D) Overlay of CD3-FITC fluorescence and background fluorescence of cells within the gate. Panels E-H: SUP-T3 cells treated with 7-AAD as described in Alternate Protocol 2 and fixed and permeabilized as described in Basic Protocol 2. Cells were stained with either mouse IgG1-FITC control antibody or CD3-FITC. (E) FSC versus SSC dot plot. (F) FSC versus 7-AAD fluorescence dot plot with the gate set around live cells. (G) Mouse IgG1-FITC (background) fluorescence of cells within the gate. (H) Overlay of CD3-FITC fluorescence and background fluorescence of cells within the gate.

    View Image

Literature Cited

 Literature Cited
    Aiello, A., Delia, D., Borrello, M.G., Biassoni, D., Giardini, R., Fontanella, E., Pezzella, F., Pulford, K., Pierotti, M., and Porta, G.D. 1992. Flow cytometric detection of the mitochondrial bcl-2 protein in normal and neoplastic human lymphoid cells. Cytometry 13:502-509.
    Anderson, P., Blue, M.L., O'Brian, C., and Schlossman, S.F. 1989. Monoclonal antibodies reactive with the T cell receptor chain: Production and characterization using a new method. J. Immunol. 143:1899-1904.
    Bauer, K.D. and Jacobberger, J.W. 1994. Analysis of intracellular proteins. Methods Cell Biol. 41:352-376.
    Blattler, W.A., Kuenzi, B.S., Lambert, J.M., and Senter, P.D. 1985. New heterobifunctional protein cross-linking reagent that forms an acid-labile link. Biochemistry 24:1517-1524.
    Clevenger, C.V. and Shankey, T.V. 1993. Cytochemistry II: Immunofluorescence measurements of intracellular antigens. In Clinical Flow Cytometry (K.D. Bauer, R.E. Duque, and T.V. Shankey, eds.) pp. 157-175. Williams and Wilkins, Baltimore.
    Clevenger, C.V., Bauer, K.D., and Epstein, A.L. 1985. A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry. Cytometry 6:208-214.
    Duncan, R.J.S., Weston, P.D., and Wrigglesworth, R. 1983. A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132:68-73.
    Fetterhoff, T.J., Holland, S.P., and Wile, K.J. 1993. Fluorescent detection of nonviable cells in fixed cell preparations. Cytometry 14:27.
    Franek, K.J., Wolcot, F.R., and Chervenak, R. 1994. Reliable method for the simultaneous detection of cytoplasmic and surface CD3 expression by murine lymphoid cells. Cytometry 17:224-236.
    Hashida, S., Imagawa, M., Inoue, S., Ruan, K.-H., and Ishikawa, E. 1984. More useful maleimide compounds for the conjugation of Fab¢ to horseradish peroxidase through thiol groups in the hinge. J. Appl. Biochem. 6:56-63.
    Jacob, M.C., Favre, M., and Bensa, J.-C. 1991. Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry. Cytometry 12:550-558.
    Jacobberger, J.W. 1989. Cell cycle expression of nuclear proteins. In Flow Cytometry: Advanced Research and Clinical Applications, Vol. 1 (A. Yen, ed.) pp. 305-326. CRC Press, Boca Raton Fla.
    Kitagawa, T., Shimozono, T., Aikawa, T., Yoshida, T., and Nishimura, H. 1981. Preparation and characterization of hetero-bifunctional cross-linking reagents for protein modification. Chem. Pharm. Bull. 29:1130-1135.
    Lanier, L.L. and Warner, N.L. 1981. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J. Immunol. Methods 47:15-30.
    Levelt, C.N. and Eichmann, K. 1994. Streptavidin-Tricolor is a reliable marker for nonviable cells subjected to permeabilization or fixation. Cytometry 15:84-86.
    Loor, F., Forni, L., and Pernis, B. 1972. The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulin. Eur. J. Immunol. 2:203-212.
    Mann, G.L., Dyne, M., and Musgrave, E.A. 1987. Immunofluorescence quantification of ribonuclease reductase M1 subunit and correlation with DNA content by flow cytometry. Cytometry 8:509-517.
    Mishell, B.B. and Shiigi, S.M. 1980. Modification and use of antibodies to label cell surface antigens. In Selected Methods in Cellular Immunology, pp. 287-304. W.H. Freeman, New York.
    Parham, P. 1983. On the fragmentation of monoclonal IgG1, IgG2a, IgG2b from BALB/c mice. J. Immunol. 131:2895-2902.
    Porter, R.R. 1959. The hydrolysis of rabbit -globulin and antibodies with crystalline papain. Biochem. J. 73:119-126.
    Puchtler, H. and Meloan, S.N. 1985. On the chemistry of formaldehyde fixation and its effect on immunohistochemical reactions. Histochemistry 82:201-204.
    Rabinovitch, P.S., Torres, R.M., and Engel, D. 1986. Simultaneous cell cycle analysis and two-color immunofluorescence using 7-amino-actinomycin D and single laser excitation: Applications to study of cell activation and the cell cycle of murine Ly-1 B cells. J. Immunol. 136:2769-2775.
    Riedy, M.C., Muirhead, K.A., Jensen, C.P., and Stewart, C.C. 1991. Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations. Cytometry 12:133-139.
    Sander, B., Andersson, J., and Andersson, U. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol. Rev. 119:65-93.
    Schmid, I., Uittenbogaart, C.H., and Giorgi, J.V. 1991. A gentle fixation and permeabilization method with combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry 12:279-285.
    Schmid, I., Krall, W.J., Uittenbogaart, C.H., Braun, J., and Giorgi, J.V. 1992. Dead cell discrimination with 7-aminoactinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13:204-208.
    Schroff, R.W., Bucana, C.D., Klein, R.A., Farell, M.M., and Morgan, A.C. 1984. Detection of intracytoplasmic antigens by flow cytometry. J. Immunol. Methods 70:167-177.
    Storek, J., Schmid, I., Ferrara, S., and Saxon, A. 1992. A novel B cell stimulation/proliferation assay using simultaneous flow cytometric detection of cell surface markers and DNA content. J. Immunol. Methods 151:261-267.
    Tanimori, H., Ishikawa, F., and Kitagawa, T. 1983. A sandwich enzyme immunoassay of rabbit immunoglobulin G with an enzyme labeling method and a new solid support. J. Immunol. Methods 62:123-131.
    Taylor, R.B., Duffus, W.P.H., Raff, M.C., and de Petris, S. 1971. Redistribution and pinocytosis of lymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody. Nature New Biol. 233:225-229.
    Titus, J.A., Haugland, R., Sharrow, S.O., and Segal, D.M. 1982. Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J. Immunol. Methods 50:193-204.
 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 Alternate Protocol 1 and shows examples of its application.

    Schmid et al., 1991. See above.

Describes Basic Protocol 2 and provides examples of its application.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.