Effect of red cell lysis protocols on the expression of rat peripheral blood lymphocyte subset and activation...

The effect of ammonium chloride and a commercial red cell lysing agent (Erythrolyse) on cell subset (CD4, CD8, B cell common leucocyte antigen) and activation (p55IL-2R [OX39], MHC class II) antigen expression by freshly isolated and in vitro activated rat peripheral blood lymphocytes 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 chloride. The expression of cell subset and MHC class II antigens was unaffected by either of the treatment protocols. Density gradient separation of freshly isolated cells had variable effects on antigen expression. In some cases up to a two-fold increase in p55IL-2R expression was observed, whilst in others there was a reduced receptor expression. Similarly, MHC class II antigen expression was either increased or reduced following density gradient separation. IL-2R expression on cells activated in vitro with the mitogen concanavalin A (95 +/- 2% IL-2R+) was unaffected by ammonium chloride lysis. Incubation of freshly isolated lymphocytes with p55IL-2R antibody prior to ammonium chloride treatment did not prevent the reduction in receptor expression. These findings suggest that ammonium chloride lysis of freshly isolated rat peripheral blood prior to IL-2R expression analysis should be avoided.