CD38 expression is associated with elevated basal Ca²⁺levels. MEC1-GFP, MEC1-CD38H, and primary CLL cells with varying CD38 expression levels (n = 17 patient samples, analyzed in 3 independent experiments) were incubated with the ratiometric Ca²⁺ indicator Indo-1 and analyzed by flow cytometry. The basal fluorescence level was recorded for 60 seconds before stimulation with CCL21 (100 ng/mL) for a further 120 seconds. (A,C) Representative bivariate plot of the median of fluorescent intensity ratio (Indo-1 emission wavelengths: 420/510 nm) against time; red boxes indicate the basal fluorescence level. (B,D) Peak values of the median basal fluorescence intensity were obtained using FlowJo Kinetic statistic tool. Horizontal bars in panel D indicate mean values of the fluorescence intensity peak calculated in each experiment for CD38^low (green) and CD38^high (red) samples. Significant differences between the 2 experimental groups were determined using a 2-tailed Student t test.