Immunophenotyping

C.C. Stewart1, S.J. Stewart1

1 Roswell Park Cancer Institute, Buffalo, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.2
DOI:  10.1002/0471142956.cy0602s00
Online Posting Date:  May, 2001
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Abstract

This unit presents basic techniques for immunophenotyping by flow cytometry, direct using a conjugated monoclonal antibody and indirect using an unconjugated primary antibody followed by a conjugated secondary antibody. Combinations of these methods are described for two‐, three‐, and four‐color staining. Analysis of data acquired from cells stained by these procedures is detailed. A procedure is given for the detection of the location of nonviable cells so that they can be gated out of the analysis.

Keywords: flow cytometry; immunophenotyping; indirect staining; direct staining; multicolor staining; immunophenotypic analysis.

     
 
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Table of Contents

  • Basic Protocol 1: Basic Indirect Staining
  • Alternate Protocol 1: Indirect Staining Using Biotinylated or Hapten‐Conjugated Antibody
  • Alternate Protocol 2: Direct Staining Procedure
  • Basic Protocol 2: Two‐Color Immunophenotyping Using Unconjugated Primary Antibody/Fluorochrome‐Conjugated Secondary Antibody in Combination with Biotinylated Antibody
  • Alternate Protocol 3: Two‐Color Immunophenotyping Using Biotinylated Antibody in Combination with Hapten‐Conjugated Antibody
  • Alternate Protocol 4: Two‐Color Immunophenotyping Using Biotinylated Antibody in Combination with Directly Conjugated Antibody
  • Alternate Protocol 5: Three‐or Four‐Color Immunophenotyping Using Unconjugated Antibody, Biotinylated Antibody, and Directly Conjugated Antibody
  • Alternate Protocol 6: Three‐ or Four‐Color Immunophenotyping Using Biotinylated, Hapten‐Conjugated, and Directly Conjugated Antibodies
  • Alternate Protocol 7: Three‐ or Four‐Color Immunophenotyping Using Biotinylated Antibody and Directly Conjugated Antibodies
  • Alternate Protocol 8: Three‐ or Four‐Color Immunophenotyping Using Unconjugated Primary Antibody and Biotinylated Secondary Antibody in Combination with Directly Conjugated Antibodies
  • Alternate Protocol 9: Three‐ or Four‐Color Immunophenotyping Using a Combination of Directly Conjugated Antibodies
  • Support Protocol 1: EMA Procedure for Detecting Nonviable Cells in a Cell Population to be Fixed
  • Data Analysis for Flow Cytometric Immunophenotyping
  • Support Protocol 2: Marker Approach Using Population Gate
  • Support Protocol 3: Marker Approach Using Cell Gate
  • Support Protocol 4: Determining Viable Cells
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Basic Indirect Staining

  Materials
  • 3 mg/ml normal goat IgG
  • 5–10 × 106 cell/ml target cell suspension in PBS ( appendix 2A)
  • Unconjugated monoclonal antibody against cell‐surface antigen of interest, appropriately titered (unit 4.1)
  • Isotype control: myeloma protein of appropriate isotype
  • Erythrocyte‐lysing solution (see recipe), 4°C
  • Fluorochrome‐conjugated polyclonal goat anti–mouse IgG F(ab′) 2, appropriately titered
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2% formaldehyde (see recipe)
  • Centrifuge and rotor capable of 2000 × g, refrigerated

Alternate Protocol 1: Indirect Staining Using Biotinylated or Hapten‐Conjugated Antibody

  • 3 mg/ml normal mouse IgG
  • Biotinylated or hapten‐conjugated monoclonal antibody against cell‐surface antigen of interest, appropriately titered (unit 4.1)
  • Isotype control: biotinylated or hapten‐conjugated myeloma protein
  • Fluorochrome‐conjugated goat anti‐hapten F(ab′) 2or fluorochrome‐conjugated streptavidin, appropriately titered

Alternate Protocol 2: Direct Staining Procedure

  • 3 mg/ml normal mouse IgG
  • Fluorochrome‐conjugated monoclonal antibody against cell‐surface antigen of interest, appropriately titered (unit 4.1)
  • Isotype control: fluorochrome‐conjugated myeloma protein

Basic Protocol 2: Two‐Color Immunophenotyping Using Unconjugated Primary Antibody/Fluorochrome‐Conjugated Secondary Antibody in Combination with Biotinylated Antibody

  Materials
  • 3 mg/ml normal goat IgG
  • 5–10 × 106 cell/ml target cell suspension
  • Unconjugated monoclonal antibody against cell‐surface antigen of interest, appropriately titered (unit 4.1)
  • Isotype control: unconjugated myeloma protein
  • Erythrocyte‐lysing solution (see recipe)
  • Fluorochrome‐conjugated polyclonal goat anti–mouse IgG F(ab′) 2, appropriately titered
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 3 mg/ml normal mouse IgG
  • Biotinylated monoclonal antibody against a second cell‐surface antigen of interest, appropriately titered (unit 4.1)
  • Isotype control: biotinylated myeloma protein
  • Fluorochrome‐conjugated streptavidin, of concentration such that appropriate quantity is contained in 10 µl
  • 2% formaldehyde (see recipe)
  • Centrifuge and rotor capable of 2000 × g, refrigerated

Alternate Protocol 3: Two‐Color Immunophenotyping Using Biotinylated Antibody in Combination with Hapten‐Conjugated Antibody

  • 3 mg/ml normal mouse IgG
  • Biotinylated monoclonal antibody against one cell‐surface antigen of interest and hapten‐conjugated monoclonal antibody against a second antigen, appropriately titered (unit 4.1)
  • Isotype control: biotinylated and hapten‐conjugated myeloma protein
  • Fluorochrome‐conjugated streptavidin, of concentration such that appropriate quantity is contained in 10 µl
  • Fluorochrome‐conjugated monoclonal antibody, of concentration such that appropriate quantity is contained in 10 µl

Alternate Protocol 4: Two‐Color Immunophenotyping Using Biotinylated Antibody in Combination with Directly Conjugated Antibody

  • 3 mg/ml normal mouse IgG
  • Biotinylated monoclonal antibody against one cell‐surface antigen of interest and fluorochrome‐conjugated monoclonal antibody against a second antigen, appropriately titered (unit 4.1)
  • Isotype control: biotinylated and directly conjugated myeloma proteins
  • Fluorochrome‐conjugated streptavidin, of concentration such that appropriate quantity is contained in 10 µl

Alternate Protocol 5: Three‐or Four‐Color Immunophenotyping Using Unconjugated Antibody, Biotinylated Antibody, and Directly Conjugated Antibody

  • One or two fluorochrome‐conjugated antibodies against cell‐surface antigens of interest, appropriately titered (unit 4.1)

Alternate Protocol 6: Three‐ or Four‐Color Immunophenotyping Using Biotinylated, Hapten‐Conjugated, and Directly Conjugated Antibodies

  • 3 mg/ml normal mouse IgG
  • Hapten‐conjugated antibody against cell‐surface antigen of interest, appropriately titered (unit 4.1)
  • One or two fluorochrome‐conjugated monoclonal antibodies against cell‐surface antigens of interest, appropriately titered (unit 4.1)
  • Isotype control: biotinylated, hapten‐conjugated, and directly conjugated myeloma proteins
  • Fluorochrome‐conjugated streptavidin, of concentration such that appropriate quantity is contained in 10 µl
  • Fluorochrome‐conjugated anti‐hapten monoclonal antibody, of concentration such that appropriate quantity is contained in 10 µl

Alternate Protocol 7: Three‐ or Four‐Color Immunophenotyping Using Biotinylated Antibody and Directly Conjugated Antibodies

  • 3 mg/ml normal mouse IgG
  • Biotinylated monoclonal antibody against cell‐surface antigen of interest, appropriately titered (unit 4.1)
  • Isotype control: biotinylated and fluorochrome‐conjugated myeloma proteins
  • Two or three fluorochrome‐conjugated monoclonal antibodies against cell‐surface antigens of interest, appropriately titered (unit 4.1)

Alternate Protocol 8: Three‐ or Four‐Color Immunophenotyping Using Unconjugated Primary Antibody and Biotinylated Secondary Antibody in Combination with Directly Conjugated Antibodies

  • Biotinylated polyclonal goat anti–mouse IgG F(ab′) 2 (Caltag Labs)
  • Isotype control: unconjugated and fluorochrome‐conjugated myeloma proteins
  • Fluorochrome‐conjugated streptavidin, of concentration such that appropriate quantity is contained in 10 µl
  • Two or three fluorochrome‐conjugated monoclonal antibodies against cell‐surface antigens of interest, appropriately titered (unit 4.1)

Alternate Protocol 9: Three‐ or Four‐Color Immunophenotyping Using a Combination of Directly Conjugated Antibodies

  • 3 mg/ml normal mouse IgG
  • Fluorochrome‐conjugated monoclonal antibodies against cell‐surface antigens of interest, appropriately titered (unit 4.1)
  • Isotype control: fluorochrome‐conjugated myeloma proteins

Support Protocol 1: EMA Procedure for Detecting Nonviable Cells in a Cell Population to be Fixed

  Materials
  • Cell suspension for analysis
  • Erythrocyte‐lysing solution (optional; see recipe)
  • EMA working solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2% formaldehyde (see recipe)
  • 12 × 75–mm test tubes
  • Centrifuge and rotor capable of 2000 × g
  • Fluorescent desk lamp
  • White pan
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Figures

Videos

Literature Cited

Literature Cited
   Riedy, M.C., Muirhead, K.A., Jensen, C.P., and Stewart, C.C. 1991. The use of a photolabeling technique to identify nonviable cells in fixed homogenous or heterologous cell populations. Cytometry 12:133‐139.
Key References
   Stewart, C.C. and Stewart, S.J. 1994. Cell preparation for the identification of leukocytes. In Methods in Cell Biology (Z. Darzynkiewicz, J. Robinson, and H. Crissman, eds.) pp. 39‐60. Academic Press, New York.
  Provides additional information on preparing cells.
   Stewart, C.C. and Stewart, S.J. 1994. Multiparameter analysis of leukocytes by flow cytometry. In Methods in Cell Biology (Z. Darzynkiewicz, J. Robinson, and H. Crissman, eds.) pp. 61‐79. Academic Press, New York.
  Provides additional information on instrument setup, data acquisition, and data analysis.
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