FIGURE 8.

Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD (A, C, D) for 15 min or filipin-III (B) for 30 min at 37 °C. For cholesterol repletion (C), cells were first incubated with MβCD and then washed and treated with 0, 1, 2, or 4 mm cholesterol for 15 min at 37 °C. Cell lysis was activated by treatment with a sublytic dose of anti-K562 antibodies and NHS (or HIS as control) for 60 min at 37 °C. D, cells were first incubated with 0, 3, or 6 mm MβCD, washed and incubated with 200 or 400 units of SLO in DTT or DTT alone (0.4 mm) as control, for 30 min at 37 °C. Cell lysis was determined by trypan blue uptake. All results are representative of four independent experiments. E, K562 cells were pretreated with 0, 3 or 6 mm of MβCD for 15 min at 37 °C. Then, the cells were treated with a sublytic dose of anti-K562 antibodies and C9D-NHS supplemented with C9-AF488 for 10 min at 37 °C. Next, the cells were washed, incubated for 20 min at 37 °C, and analyzed under a confocal microscope. The amount of intracellular C9-AF488 was quantified with the Zeiss 410 LSM software in 100 randomly selected cell images (Relative fluorescence intensity; R.F.I.). *, p < 0.05; **, p < 0.01; ***, p < 0.001.