Anti-IgM (HB57 dex)–mediated membrane proximal events. (A) Induction of Ca²⁺ flux by B-CLL cells. Ratiometric analysis of Indo-1 fluorescence in its Ca-bound to free state indicative of intracellular [Ca²⁺] was performed over a 15-minute period after activation with various reagents: PMA + ionomycin, goat anti–human-μ, or HB57dex. In all experiments, the ratio of Ca²⁺ mobilization in response to a signal to unstimulated was calculated over a 5-minute period (vertical dotted line). The figure shows representative tracings from 1 B-CLL case included in optimization studies. (B) Fold induction of Ca²⁺ flux by BCR stimulation using HB57-dex compared with baseline levels in 16 U-CLL and 15 M-CLL patients. Baseline levels were established by acquiring data from unstimulated cells for 30 seconds before addition of the stimulants. Statistical significance of the comparisons was evaluated using the paired t test. Differences were not significant. (C-E) BCR-mediated phosphorylation of Akt, Erk, and MAPK. Fold change in mean fluorescence intensity acquired by flow cytometry elicited by HB57-dex over unstimulated controls in all cases (2 scattergrams on the left); scattergrams on the right represent values obtained in cases segregated by IGHV gene mutations. (C) pAkt. (D) pErk. (E) p-p38MAPK. Statistical significance of the comparisons was evaluated using the paired t test. Differences were not significant.