Simultaneous cytofluorometric analysis for the expression of cytoplasmic antigens and DNA content in CD3- human thymocytes

M. R. Zocchi, F. Marelli, A. Poggi

Research output: Contribution to journalArticle

Abstract

We describe a method of two-color immunofluorescence staining which allows the simultaneous analysis of both cytoplasmic antigens and cell entry into the S/G2/M cell cycle phases. This analysis was performed on CD3-activated thymocytes obtained from either highly purified CD1-CD3-CD4-CD8- cells or fresh thymus cell suspensions, stimulated with low doses of phorphol-12 myristate-13 acetate (0.5 ng/ml) and interleukin-2. On the 14th day under these culture conditions about 90% of thymocytes did not express CD3 antigen on the cell surface. CD3- cells were further purified by cell sorting, fixed in paraformaldehyde, and permeabilized with Nonidet-P40. Then these thymocytes were stained by indirect immunofluorescence with monoclonal antibodies identifying T cell-specific molecules (CD3, CD2, CD28, TCRα/β, and TCR γ/δ) and analyzed for DNA content. Interestingly, both CD3 and CD28 antigens were detectable in the cytoplasm of most cells (> 80%). Further, the majority of the thymocytes which had entered the S/G2/M phases of the cell cycle (20%) expressed intracellular CD3 and CD28 molecules and reacted with the anti-β framework βF1 monoclonal antibody. The relationship between the appearance of CD3 and other T cell markers in the cytoplasm, the cell cycle entry, and the thymocyte development is discussed.

Original languageEnglish
Pages (from-to)883-887
Number of pages5
JournalCytometry
Volume11
Issue number8
Publication statusPublished - 1990

Keywords

  • CD3 thymocytesf
  • Cell Cycle
  • Cytoplasmic fluorescence

ASJC Scopus subject areas

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

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