A Method for Counting Phytohemagglutinin-Stimulated Lymphocytes

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Abstract
A technic is described for accurately enumerating the number of viable. PHA-stimulated lymphocytes in culture. This technic is both simple and fast. The technic depends upon the elimination of debris by means of the proteolytic enzyme, pronase, and the removal of cell cytoplasm by cetrimide leaving clean, intact nuclei which can be counted and sized. Serially determined absolute lymphocyte counts in cultures with phytohemagglutinin are reported. These show that there is a net gain in both cellularity and number of cells incorporating tritiated thymidine and that there is logarithmic growth throughout the 72-hour culturing interval. Quantitative studies of this lymphocyte response as a function of PHA concentration are in progress.