EFFECT OF RED-CELL LYSIS PROTOCOLS ON THE EXPRESSION OF RAT PERIPHERAL-BLOOD LYMPHOCYTE SUBSET AND ACTIVATION ANTIGENS

The effect of ammonium chloride and a commercial red cell lysing agent (Erythrolyse) on cell subset (CD4, CD8, B cell common leucocyte antig en) and activation (p55IL-2R [OX39], MHC class II) antigen expression by freshly isolated and in vitro activated rat peripheral blood lympho cytes was compared. Freshly isolated cells treated with the commercial lysing agent Erythrolyse were 42 +/- 12% p55IL-2R+, whereas p55IL-2R expression was not detected following red cell lysis with ammonium chl oride. The expression of cell subset and MHC class II antigens was una ffected by either of the treatment protocols. Density gradient separat ion of freshly isolated cells had variable effects on antigen expressi on. In some cases up to a two-fold increase in p55IL-2R expression was observed, whilst in others there was a reduced receptor expression. S imilarly, MHC class II antigen expression was either increased or redu ced following density gradient separation. IL-2R expression on cells a ctivated in vitro with the mitogen concanavalin A (95 +/- 2% IL-2R+) w as unaffected by ammonium chloride lysis. Incubation of freshly isolat ed lymphocytes with p55IL-2R antibody prior to ammonium chloride treat ment did not prevent the reduction in receptor expression.These findin gs suggest that ammonium chloride lysis of freshly isolated rat periph eral blood prior to IL-2R expression analysis should be avoided.