QUANTITATION OF LYMPHOCYTE PROLIFERATIVE RESPONSE TO ALLOGENEIC CELLS AND PHYTOHEMAGGLUTININ BY FLOW CYTOFLUOROMETRY .2. COMPARISON WITH(C-14) THYMIDINE INCORPORATION

Lymphocyte [human] blast transformation in response to stimulation by phytohemagglutinin (PHA) or by allogeneic cells in 1-way mixed lymphocyte culture was quantitated using acridine orange (AO) staining and flow cytofluorometry. The method involves staining of fixed cells with AO in such a manner that RNA stains metachromatically red and DNA stains green. Pyroninophillic blast cells with increased cytoplasmic RNA are identified by their high AO red fluorescence per cell, and can be counted by flow cytofluorometry in stimulated and unstimulated cultures. A comparison was made of the proliferative response of stimulated normal peripheral blood lymphocytes (PBL) as determined by AO cytofluorometry and by incorporation of [14C]thymidine into parallel microcultures. The 2 methods show a similar proliferative response to varying concentrations of PHA and to increasing strength of mixed lymphocyte interaction. AO cytofluorometry can separately and simultaneously measure the increase in number of stimulated cells (proliferative response) and per-cell RNA synthesis (nonproliferative response) in response to immunologic stimulation.