Figure 6. Cholesterol Is Necessary for CD20 Binding and Rituximab-Mediated CDC.

Raji cells were incubated with either diluent or lovastatin (10 μM for 48 h [A and B]), simvastatin (10 μM for 48 h [C and D]) or MβCD (5 mg/ml for 30 min [E and F]). Water-soluble cholesterol (chol; 0.2 mg/ml) was added for 2 h prior to flow cytometry analysis (A, E) or addition of 10 μg/ml rituximab and 10% human AB serum (B and F). Mevalonic acid (MA) was added together with simvastatin at a 200 μM concentration (C and D). Then for flow cytometry studies (A, C, and E), 1 × 10⁶/ml of cells were incubated with saturating amounts of FITC-conjugated anti-CD20 mAb (B9E9) or IgG1 (isotype control) for 30 min at room temperature in the dark. Cell viability was measured in a MTT assay (B, D, and F). The survival of cells is presented as percentage of corresponding diluent- or statin-pretreated cells without rituximab. *p < 0.001 (two-way Student's t-test) as compared to statin-treated (B and D) or MβCD-treated (F) cells.