A decrease in lipid packing precedes PS exposure in B cells. (A) Lymphocytes from C57BL/10 mice were labeled with anti-CD19^APC antibody and preincubated with either 0.05 μM MC540 or AV^FITC. Histograms show (mean fluorescence units) the rate of (Ai) MC540 binding or (Aii) PS exposure (binding of AV) by CD19⁺ B cells following stimulation with 3 μM (red lines), 2 μM (blue lines), or 1 μM (green lines) calcimycin. (Bi) Lymphocytes from C57BL/10 and NOD mice were labeled with anti-CD19^APC and anti-CD19^PERCP antibodies, respectively, mixed, and preincubated with 0.05 μM MC540. Panels show density plots of the rate of decrease in lipid packing (increased uptake of MC540) by B cells from C57BL/10 (left panel) and from NOD mice (center panel) in the same tube following stimulation with 2 μM calcimycin (arrows). The right panel shows an alternative plot of the same data plotted as the percentage of cells binding high levels of MC540 as a function of time. Red lines, NOD B cells; blue lines, C57BL/10 B cells. (Bii) Histograms of MC540 binding by the B-cell populations shown in panel Bi, gated at t = 0 seconds and t = 400 seconds. Red lines, NOD B cells; blue lines, C57BL/10 B cells. (C) Results of 3 independent experiments equivalent to that in panel Bi but with lymph node cells derived from NOD (red lines) and DBA/2 (green lines) mice. (D) Calcium uptake in stimulated B cells. (Di) Lymphocytes from the C57BL/10 (red line) and NZW (blue line) mice were labeled with anti-CD19^APC and anti-CD19^PERCP antibodies respectively, mixed, incubated with 0.25 μM of the calcium-sensitive indicator Fluo-4AM for 10 minutes, washed, and stimulated with 0.2 μM calcimycin (arrow). Plots show Ca²⁺ uptake as a function of time. (Dii) Shows an equivalent experiment to that in panel Di but with lymph node cells derived from DBA/2 (red line) and NZW (blue line) mice.