EVALUATION OF DIFFERENT FIXATION-PERMEABILIZATION METHODS FOR SIMULTANEOUS DETECTION OF SURFACE, CYTOPLASMIC MARKERS AND DNA ANALYSIS BY FLOW-CYTOMETRY IN SOME HUMAN HEMATOPOIETIC-CELL LINES

Different fixation/permeabilization methods were investigated for thei r convenience for simultaneous detection of membrane marker/DNA staini ng or cytoplasmic marker/DNA staining by flow cytometry. Nine differen t methods were employed. The expression of membrane marker CD20 and cy toplasmic marker CD22 on BJAB and DAUDI cells and cytoplasmic CD3 on M OLT4 cells was measured. Optimal methods were those that combine paraf ormaldehyde and saponin (for membrane CD20/DNA staining and cytoplasmi c CD3/DNA staining) or buffered formaldehyde-acetone (for membrane CD2 0/DNA staining and for cytoplasmic CD22/DNA staining). Special interes t was focused on proliferation marker CD71 and nuclear antigen Ki67. W e investigated CD71 in MOLT4 cells and Ki67 in MOLT4, DAUDI and BJAB c ells. Both of these markers are closely associated with proliferation rate. The optimal method for the detection of Ki67/DNA staining combin es paraformaldehyde with Tween 20.