Simultaneous cytofluorometric analysis for the expression of cytoplasmic antigens and DNA content in CD3- human thymocytes.
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, TCR alpha/beta, and TCR gamma/delta) and analyzed for DNA content. Interestingly, both CD3 and CD28 antigens were detectable in the cytoplasm of most cells (greater than 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-beta framework beta F1 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.
Zocchi
M R
MR
Laboratorio Immunoterapia Adottiva, Istituto Scientifico San Raffaele, Milan, Italy.
Marelli
F
F
Poggi
A
A
D015703:Antigens, CD / Q000032:analysis*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D017252:CD3 Complex; D002454:Cell Differentiation / Q000276:immunology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D002648:Child; D003593:Cytoplasm / Q000276:immunology; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry; D006801:Humans; D007376:Interleukin-2 / Q000502:physiology; D011948:Receptors, Antigen, T-Cell / Q000032:analysis*; D013601:T-Lymphocytes / Q000166:cytology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology / Q000254:growth & development*; D013997:Time Factors
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D004247:DNA; D013755:Tetradecanoylphorbol Acetate