mAb ligation of CD38 induces Ca²⁺ waves in purified B-CLL cells. B-CLL cells purified from patients 1, 8, 21, and 27 were loaded with the fluorescent indicator Fluo 3-am and preincubated (10 minutes at room temperature) with an agonistic anti-CD38 mAb (A,C,E,G). The cells were then washed and analyzed continuously at 37°C using a FACSort, while a RαMIg was added as cross-linker (arrows) 10 seconds after starting the analysis. IgM and IgD ligation using specific polyclonal antibodies induced Ca²⁺ responses, as expected, whereas no synergy was observed when adding the anti-CD38 mAb (I,K). The same cells were cultured for 3 days in the presence of 100 IU/mL IL-2 and re-evaluated for Ca²⁺ mobilization on CD38 cross-linking (B,D,F,H), as well as for IgM (J) and IgD (L). A nonagonistic anti-CD38 mAb was unable to mobilize Ca²⁺ ions (M). Appropriate dye loading by the cells was checked by the addition of the ionophore A23187 (N, arrow). Data are presented as density plot of the shift in the Fluo 3-am fluorescence (y-axis) over a 512-second period (x-axis).