Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry

Alan Landay, Cheryl Jennings, Meryl Forman, Robert Raynor

Research output: Contribution to journalArticlepeer-review


Dual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CD8+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA‐1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CD8+ cells. The method effectively quantitated TIA‐1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system. © 1993 Wiley‐Liss, Inc.

Original languageEnglish (US)
Pages (from-to)433-440
Number of pages8
Issue number4
StatePublished - 1993
Externally publishedYes


  • digitonin
  • Intracellular antigen
  • multiparameter

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology


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