A CYTOFLUOROMETRIC METHOD FOR THE SIMULTANEOUS DETECTION OF BOTH INTRACELLULAR AND SURFACE-ANTIGENS OF APOPTOTIC PERIPHERAL LYMPHOCYTES

The aim of this study was to define a simple and reliable method to de tect simultaneously surface and intracellular antigens in apoptotic pe ripheral human lymphocytes. This approach requires a permeabilizing pr ocedure for intracellular access of mAbs, which raises the important q uestion of the influence of this procedure on parameters which identif y apoptotic cells and on the surface expression of antigens. We compar ed the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantifica tion of apoptotic lymphocytes, defined according to FSC/SSC criteria o r following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface an tigens with intracellular molecules, including Bcl-2 and cytokines (IF N gamma, TNF alpha, IL-2) was also analysed in the context of these th ree permeabilizing procedures. All the experiments were performed on P BMC from HIV-infected donors, known to undergo excessive apoptosis fol lowing short-term culture. We report that permeabilization with saponi n is the only procedure which allows: (1) the preservation of lymphocy te morphology determined by the FSC/SSC parameters; (2) the quantifica tion of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capaci ty (ABC); (4) the proper detection of intracellular membrane bound ant igens (Bcl-2) and intracellular cytokines (IFN gamma, TNF alpha, IL-2) ; (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate th at the simultaneous analysis of extracellular and intracellular antige ns in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order. (C) 1998 Published by Elsevier Science B.V. All rights reserved.