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1. Clearly, the simplest (and the least expensive) assay is analysis
of DNA/RNA content. Activation (G0/G1 transition measured based on
the increase in RNA content) is detected as early ad 14 - 24 h after
addition of the mitogen. The cell entrance to S phase (after 24 h)
can be then easily detected based on DNA content analysis. The
methodology can be found in Methods in Cell Biol., Vol 41. pp
402-420, 1994.
2. Another useful activation antigen is cyclin D3. Nonstimulated
lymphocytes are cyclin D3 negative. The appearance of cyclin D3 is
rapid, detected already 4h after addition of the mitogen, and is
specific to G0/G1 transition. Maximal expression of this cyclin is
seen between 8 and 24 h, prior to cell entrance to S phase. The
cyclin D3 antibody is commercially available (e.g. Pharmingen). Flow
cytometric detection of cyclins D2 and D3 during lymphocyte
stimulation is described in by Gong et al., in Leukemia, 9: 893-899,
1995.
Zbigniew Darzynkiewicz
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