Fluorescence measurements of fusion between human erythrocytes induced by poly(ethylene glycol)

The kinetics of poly(ethylene glycol) (PEG)-induced fusion between intact human erythrocytes was continuously monitored by a fluorescence lipid mixing method, utilizing the dequenching of the fluorescence probe, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl ] phosphatidylcholine (C12-NBD-PC). The steady-state fluorescence intensity was detected from the surface of cells in a monolayer on an alcian blue-coated glass coverslip. The relief of fluorescence self-quenching after fusion between C12-NBD-PC labeled and unlabeled intact erythrocytes was measured. The extent of fluorescence dequenching was normalized based on the measured concentration of probes in membranes, the projected partial dequenching due both to dilution by intercellular fusion, and the dilution between the inner and outer leaflets of membranes (flip-flop). There was no significant increase in fluorescence intensity during PEG treatment of 5 min, at 4 degrees C. Intensity increased immediately after the dilution of PEG, and reached saturation in 30 min. The efficiency of fusion increased with the increasing of PEG concentrations. Only 4% enhancement of saturated relative fluorescence intensity was detected in 25 wt% PEG-induced cell fusion; 23% enhancement in 30 wt%; and 66% enhancement in 35 wt%. The transfer of fluorescent probes between membrane bilayer leaflets (flip-flop) was also monitored during the fusion process. Flip-flop was monitored in confluent monolayers as well as in isolated cells. There was no significant spontaneous flip-flop within 30 min of dilution. The relative fluorescence intensity enhancement contributed by the dilution of probes between fused labeled and unlabeled cells (at a 1:1 ratio) was found to account for only 39% of the observed final dequenching, whereas the contribution by flip-flop associated with cell fusion was found to account for 9%, and flip-flop without fusion contributed approximately 18%. A portion of the flip-flop is a consequence of hemolysis. Therefore, fluorescence dequenching measurements of fusion of whole cells must be interpreted with caution.