FIGURE 1.

Cdt holotoxin association with Jurkat cells is dependent upon the presence of cholesterol. Jurkat cells were exposed to medium (panels A and D) or 5 mm MβCD (panels B and E (Sigma)) for 30 min. Cells were washed, and some of the MβCD-treated cells were incubated with cholesterol-saturated MβCD (0.5 mm) for 30 min (panels C and F). Cells were incubated with Cdt holotoxin (2 μg/ml) for 1 h, washed, and treated with control murine IgG (data not shown) or anti-CdtC monoclonal antibody conjugated to Alexafluor 488. Jurkat cells were then analyzed by flow cytometry (panels A–C), or images of cells were taken on a Bio-Rad Radiance 2100 Confocal Microscope (Bio-Rad); representative images are shown in panels D–F. Results are representative of three experiments. The relative level of cholesterol in 10⁷ Jurkat cells was compared by semiquantitative TLC. Cholesterol was identified based on calculated Rf values and color after detection by charring with sulfuric acid/EtOH (1:1 vol:vol). The intensity of cholesterol for each sample was compared with the intensity of known standards using digital densitometry; values were 11.8, 0.37, and 20.7 μg cholesterol/10⁷ cells for control, MβCD-treated, and cholesterol-saturated MβCD-treated cells, respectively.