Analysis of lymphocyte activation and proliferation by video microscopy and digital imaging

Video microscopy and digital imaging were used as a noninvasive method to quantitatively analyze lymphocyte activation and proliferation. This method takes advantage of the fact that upon activation lymphocytes blast and become significantly larger before proliferating. The mean cell sizes of T lymphocytes in an activation kinetics assay were measured by digital image analysis and compared to [3H]-thymidine incorporation of cells under the same treatment. An increase in cell size was observed before [3H]-thymidine incorporation; therefore the digital imaging assay is more sensitive in determining the earliest time-point of activation. Also, the digital imaging assay was comparable to the [3H]-thymidine incorporation assay in providing information about the extent and rates of T lymphocyte proliferation. Cellular DNA was stained with propidium iodide to show that the larger blasting cells in the population of activated T lymphocytes were indeed the cells that accounted for the increase in DNA synthesis and thus an increase in cell size can be correlated with activation.