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

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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 phorbol‐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 cell (2/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.