Enzymatic Amplification Staining for Cell Surface Antigens

David Kaplan1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.14
DOI:  10.1002/0471142956.cy0614s23
Online Posting Date:  February, 2003
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Abstract

Many molecules of biological significance function at very low levels and are difficult to detect with the standard staining procedures for flow cytometric analysis. This unit describes a technology for cell staining that enhances the specific fluorescence signal by as much as 100‐fold. The system is based on the enzyme‐catalyzed deposition of a tagged molecule. Enzymatic amplification staining is qualitatively different from the inclusion of additional layers of binding molecules because background fluorescence levels are not increased along with the specific signal. The technique is compatible with multicolor staining. An alternate protocol explains the performance of multiple amplifications on the same cell population by adding a peroxide incubation.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: EAS for Cell Surface Molecules
  • Alternate Protocol 1: Multicolor Staining with Amplification of a Single Marker with EAS
  • Alternate Protocol 2: Multiple Amplifications with EAS on a Single Cell Population
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: EAS for Cell Surface Molecules

  Materials
  • Cells in a single‐cell suspension in phosphate buffered saline (PBS), pH 7.2 to 7.4 ( appendix 2A for PBS)
  • Staining buffer: PBS, pH 7.2 to 7.4, with 2% fetal bovine serum, sterile filtered
  • Unconjugated, biotinylated, or fluorescein isothiocyanate (FITC)‐conjugated antibody specific for the antigen of interest in PBS, pH 7.2 to 7.4, with 2% fetal bovine serum
  • Isotype/subtype control immunoglobulin unconjugated, biotinylated, or FITC‐conjugated in PBS, pH 7.2 to 7.4, with 2% fetal bovine serum
  • EAS kit for unconjugated, biotinylated, or FITC‐conjugated primary antibodies (Flow‐Amp Systems) containing horseradish peroxidase–conjugated secondary reagents (anti‐immunoglobulin antibody, streptavidin, or anti‐FITC antibody), amplification medium, amplification reagent, and streptavidin‐FITC
  • 12 × 75–mm polystyrene round‐bottom test tubes
  • Flow cytometer with 488‐nm excitation and filters for collection of green fluorescence

Alternate Protocol 1: Multicolor Staining with Amplification of a Single Marker with EAS

  • Monoclonal antibodies directly conjugated with fluorochromes
  • Filters for collection of fluorescence emission from fluorophores used

Alternate Protocol 2: Multiple Amplifications with EAS on a Single Cell Population

  • 1% hydrogen peroxide in PBS
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Figures

Videos

Literature Cited

Literature Cited
   Bernard, J., Treton, D., Vermot‐Desroches, C., Boden, C., Horellou, P., Angevin, E., Galanaud, P., Wijdenes, J., and Richard, Y. 2001. Expression of interleukin 13 receptor in glioma and renal carcinoma: IL13α2 as a decoy receptor for IL13. Lab. Invest. 81:1223‐1231.
   Bobrow, M., Harris, T., Shaughnessy, K., and Litt, G. 1989. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods 125:279‐285.
   Bobrow, M., Shaughnessy, K., and Litt, G. 1991. Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays. J. Immunol. Methods 137:103‐112.
   Deimann, W. 1984. Endogenous peroxidase activity in mononuclear phagocytes. Prog. Histochem. Cytochem. 15:1‐58.
   DeSombre, E., Anderson, W., and Kang, Y. 1975. Identification, subcellular localization, and estrogen regulation of peroxidase in 7,12‐dimethylbenz(a)anthracene‐induced rat mammary tumors. Cancer Res. 35:172‐179.
   Horner, A., Widhopf, G., Burger, J., Takabayashi, K., Cinman, N., Ronaghy, A., Spiegelberg, H., and Raz, E. 2001. Immunostimulatory DNA inhibits IL‐4‐dependent IgE synthesis by human B cells. J. Allergy Clin. Immunol. 108:417‐423.
   Jabara, H., Broder, S., and Geha, R. 2001. Glucocorticoids upregulate CD40 ligand expression and induce CD40L‐dependent immunoglobulin isotype switching. J. Clin. Invest. 107:371‐378.
   Kaplan, D. and Smith, D. 2000. Enzymatic amplification staining for flow cytometric analysis of cell surface molecules. Cytometry 40:81‐85.
   Kaplan, D. 2002. Flow cytometric analysis of cells from persons with HIV‐1 disease by enzymatic amplification staining. In Cellular Aspects of HIV Infection (A. Cossarizza and D. Kaplan, eds.) pp. 351‐369. John Wiley & Sons, New York.
   Kaplan, D., Meyerson, H., and Lewandowska, K. 2001a. High resolution immunophenotypic analysis of chronic lymphocytic leukemia cells by enzymatic amplification staining. Amer. J. Clin. Pathol. 116:429‐436.
   Kaplan, D., Husel, W., and Meyerson, H. 2001b. Immunophenotypic analysis with enhanced sensitivity of detection by enzymatic amplification staining. Clin. Lab. Med. 21:763‐778.
   Kaplan, D., Smith, D., Meyerson, H., Pecora, N., and Lewandowska, K. 2001c. CD5 expression by B lymphocytes and its regulation upon Epstein‐Barr virus transformation. Proc. Natl. Acad. Sci. U.S.A. 98:13850‐13853.
   Kennedy, N., Russell, J., Michail, N., and Budd, R. 2001. Liver damage by infiltrating CD8+ cells is Fas dependent. J. Immunol. 167:6654‐6662.
   Li, C., Ziesmer, S., and Lazcano‐Villareal, O. 1987. Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method. J. Histochem. Cytochem. 35:1457‐1460.
   Xinhluo, L., Bai, X., Wen, J., Gao, J., Liu, J., Lu, P., Wang, Y., Zheng, P., and Liu, Y. 2001. B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8+ T lymphocytes in vivo. J. Exp. Med. 194:1339‐1348.
Key Reference
   Kaplan, D. and Smith, D. 2000. See above.
  Validation of the EAS procedure.
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