FLOW CYTOMETRIC ANALYSIS OF CELL-KILLING BY THE JUMPER ANT VENOM PEPTIDE PILOSULIN-1

Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corr esponds to the largest allergenic polypeptide found in the venom of th e jumper ant Myrmecia pilosula. Initial experiments showed that pilosu lin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how how cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluo rochrome-conjugated antibodies, incubated with the peptide and 7-amino actinomycin D (7-AAD), and then analyzed. The effects of varying the p eptide concentration, serum concentration, incubation time, and incuba tion temperature were measured, and the cytotoxicity of pilosulin 1 wa s compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and d ead cells, respectively, It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three o r more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melit tin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than me littin; 3) both pilosulin 1 and melittin were more potent against mono nuclear leukocytes than against granulocytes; and 4) serum inhibited k illing by either peptide. Cytometry 32:268-273, 1998. (C) 1998 Wiley-L iss, Inc.