CD4⁺T cells stimulating assay by flow cytometry.

Abstract

<p><b>A and B</b>, Purified naïve CD4⁺T cells were cultured in wells with PBS, anti-CD3 mAb alone (5 µg/ml), anti-CD3 mAb (5 µg/ml), plus anti-CD28 mAb (2 µg/ml), and PHA-M (10 ng/ml) as indicated. After 3 days culturing, the expression of CD69 was assessed by flow cytometry. The percentages represented positive CD69 populations after CD4⁺T stimulation. The histogram of a representative experiment is presented in A (blank line, CD69 staining). CD4⁺T cells stimulated by anti-CD3 Ab plus anti-CD28 Ab elevated CD69 expression by 67.95%, compared with PBS (2.76%), anti-CD3 alone (26.78%) or PHA-M (31.57%), displaying the most efficient T cells proliferation. The summary of 3 independent experiments is presented in B. <b>C</b>, Cell division of CD4⁺T cell subpopulations were measured by CFSE dilution on day 3 by flow cytometric analyses of CD3/CD28 stimulating cells. (blue line, Isotype control; blank, CD69 staining).</p