A FLOW CYTOMETRIC METHOD TO ASSESS ANTIGEN-SPECIFIC PROLIFERATIVE RESPONSES OF DIFFERENT SUBPOPULATIONS OF FRESH AND CRYOPRESERVED HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS

We have used PKH26 dye, which is incorporated stably into the membrane of cells, to determine, using flow cytometry, lymphocyte proliferativ e responses to the antigen tetanus toroid in fresh and cryopreserved s amples. Measuring cell proliferation with this dye has advantages over either H-3-thymidine or Bromodeoxyuridine (BrdU). Whereas the existin g methods measure proliferation at a single time point, PKH26 gives a cumulative measure of cell proliferation. As PKH26 is incorporated int o the cell membrane, cells do not have to be permeabilised to allow dy e incorporation into a cytoplasmic compartment. Most importantly, PKH2 6 can be used in combination with monoclonal antibodies to surface mar kers on mixed populations of cells, to determine the proliferation of individual subpopulations, without the need for prior cell fractionati on. We also show that PKH26 can be used with similar efficacy in both fresh and cryopreserved samples. In addition since PKH26 is a cumulati ve measure of proliferative responses we were able to show that restim ulation of the dividing population in vitro with fresh antigen present ing cells (APC) and antigen permits characterisation of a further prol iferating cell population. The use of PKH26 dye in combination with ce ll phenotyping and measurement of cytokine production at the single ce ll level will prove a powerful tool for multiparameter analyses of cel lular responses to antigen. (C) 1998 Elsevier Science B.V. All rights reserved.