Figure 1. Altered membrane properties and reduced stability of RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL antibodies.

A. The osmotic fragility of RBC_VL (open square) compared to RBC_N (filled square) was measured by degree of hemolysis with increasing osmotic stress by incubation in NaCl (0–0.9%) determined by spectrophotometry. Data are represented as % hemolysis, are means of triplicates ± SD and representative of three independent experiments. B. Sensitization of RBC_VL for hemolysis with anti-9-O-AcSGP IgG_VL antibodies. RBC_VL (open bars) and RBC_N (filled bars) were incubated for 30 min at 37°C with increasing concentration (0.5 µg/ml–12.0 µg/ml) of anti-9-O-AcSGP IgG_VL and anti-9-O-AcSGP IgG_NHS, respectively. The extent of hemolysis was then determined by spectrophotometry. The Spectrophotometric readings of RBC_VL or RBC_N in buffer under similar condition were taken as base values and subtracted from the experimental values. Data are expressed as means ± SD of three independent experiments. C. Kinetics of the hemolysis of RBC_VL and RBC_N after sensitization with anti-9-O-AcSGP IgG_VL antibodies. RBC_VL (open squares) and RBC_N (filled squares) were incubated with equal (6.0 µg/ml) amounts of anti-9-O-AcSGP IgG_VL and anti-9-O-AcSGP IgG_NHS, respectively, for different time points (0–120 min). Base value was routinely subtracted from each experimental value as in Fig. 1B. Data are means ± SD of three independent experiments. D. Hemolysis of RBC_N (filled bars) and RBC_VL (open bars) after sensitization of RBC_VL with anti-9-O-AcSGP IgG_NHS or anti-9-O-AcSGP IgG_VL (6.0 µg/ml), respectively, or incubation with the calcium ionophore A23187 (1 µM) in Ringer solution for 30 min. Data are means ± SD of three independent experiments. E. Enhanced membrane hydrophobicity of RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL. The hydrophobicity was determined spectrofluorimetrically using ANS as indicator. RBC_VL were incubated with anti-9-O-AcSGP IgG_VL antibodies or PBS only for 30 min at 37°C. As controls, RBC_N were incubated with anti-9-O-AcSGP IgG_NHS. The cells were washed with PBS, loaded with ANS and incubated for 1 h at 37°C. The fluorescence emission spectra were recorded from 400 to 850 nm with excitation at 365 nm. The graphs are representatives of the results obtained from three independent experiments. F. Morphological changes of RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL antibodies. RBC_N and RBC_VL were not sensitized or sensitized with anti-9-O-ACSGP IgG_NHS or anti-9-O-AcSGP IgG_VL antibodies, respectively, (6.0 µg/ml) for 30 min at 37°C, and then processed for SEM as described in Materials and Methods. Representative images from three independent experiments are shown. G. Decrease of RBC_VL cell size after sensitization detected by low-angle scattering light flow cytometry (forward scatter, FCS). FCS was measured of RBC_N (upper left panel) and of RBC_VL without sensitization (upper right panel) or with sensitization with anti-9-O-AcSGP IgG_VL (2.5 µg/ml) (lower left panel) or treatment with A23187 (1.0 µM) (lower right panel). Figure 1F is excluded from this article's CC-BY license. See the accompanying retraction notice for more information.