Figure 3.

Release of intracellular calcium and IP₃. (a) Intracellular calcium flux is reduced in Vav-deficient CD4⁺ splenic T cells. The two panels at the top show flow cytometric analysis of CD4 and CD8 staining of Vav-deficient (Vav^−/−) or control (Vav^+/+) splenocytes preloaded with Indo-1 and coated with anti-CD3. Panels below show intracellular calcium concentrations in the CD4⁺ T cells (gated as shown in the CD4/CD8 plots) as a ratio of Indo-1 violet/blue fluorescence versus time. Cells were stimulated with 0.1–100 μg/ml goat anti-hamster antibody to crosslink the anti-CD3 at the time indicated by the break in the calcium trace. Adequate loading of the Vav^−/− T cells with Indo-1 was demonstrated by inducing a calcium flux with ionomycin (not shown). (b) Graph showing mean levels of IP₃ (±SEM) in CD4⁺ T cells precoated with anti-CD3 and anti-CD28 antibodies and stimulated by the addition of gαhIg (300 μg/ml).