Analysis of activated human lymphocytes membrane antigens by dual laser flow cytometry

• Published in: Japanese Journal of Clinical Immunology Vol. 6; no. 6; pp. 523 - 534
• Main Authors: Matsuo, Yoshinobu, Yokoyama, Mitsuo, Kinoshita, Katsumi, Nagata, Yoshihiko, Fujita, Kazuhiro
• Format: Journal Article
• Language: English
• Published: The Japan Society for Clinical Immunology 1983
• Subjects: combined immunofluorescent staining method; concanavalin A; dual laser flowcytometry; mitogen induced activated T cell antigens; phytohemagglutinin-P; pokeweed mitogen
• ISSN: 0911-4300; 1349-7413
• DOI: 10.2177/jsci.6.523

Cell surface antigens of normal human peripheral lymphocytes after activation with mitogenic lectins; phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) were analysed by dual laser flow cytometer. Normal human peripheral blood mononuclear cells were isolated by Ficoll-Conray sedimentation method. The cells were washed three times with RPMI-1640 medium and were resuspended into RPMI-1640 medium supplemented 10% fetal calf serum. The cell suspension was adjusted to a concentration of 1×106/ml and PHA, Con A and PWM were added to the cell suspension at an optimal concentration of the lectins respectively. The cells were then cultured at 37 C° humidified 5% CO2 atmosphere for 72 hrs. After 72 hrs, the cells were harvested, washed three times with phosphate buffered saline (PBS) and cell surface markers were analysed by direct, indirect and combined immunofluorescent staining methods. The combined staining method was carried out first, by indirect staining with tetramethyl rhodamine isothiocyanate (TRITC) conjugated goat anti-mouse IgG serum as the secondaly reagent and second, by direct staining with fluorescein isothiocyanate (FITC) conjugated T4 and T8 mouse monoclonal antibody. Activated T cell antigen recognized with anti-Tac monoclonal antibody, other T cell antigens recognized with T4, T8, OKT-9, OKT-10 monoclonal antibodies and immune associated (Ia)-like antigen recognized with I2 monoclonal antibody were markedly expressed on the cell surface of lectin stimulated normal humal peripheral lymphocytes. Through the dual laser flow cytometric analysis, all T4 positive cells exhibited the Tac antigen. The T8 positive cells also exhibited the Tac antigen but not on all cells. The Tac negative but the T8 positive and double marker cells representing both T4 and T8 positive were also demonstrated. Acute lymphoblastic leukemia antigen (CALLA) with J5, B cell antigen with Bl and myelomonocyte antigen with Mo 2 monoclonal antibody were not recognized on the distribution of two membrane surface antigens on the cells with a single procedure.