A Method for Counting Phytohemagglutinin-Stimulated Lymphocytes

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 cetrimideleaving 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 islogarithmic growth throughout the 72-hour culturing interval. Quantitativestudies of this lymphocyte response as a function of PHA concentration arein progress.

Submitted on August 10, 1966 Accepted on December 5, 1966