The effect of cross-linking the ₂-integrin subunit of mouse megakaryocytes on [Ca²⁺]_ilevels. (A) After plating of a bone marrow suspension onto poly-L-lysine–coated coverslips, cells were coated with anti-₂ MoAb for 5 minutes and then the excess was removed. Extracellular medium ([Ca²⁺]_e = 200 μmol/L) was added and a suitable cell was microinjected with FURA-2. The change in [Ca²⁺]_i in response to the addition of antihamster secondary Ab (20 μg/mL) as indicated was determined. (B) After FURA-2 injection of a suitable cell, anti-₂ MoAb was added (10 μg/mL) and the change in [Ca²⁺]_i was recorded. In (C), similar experiments were performed after the addition of secondary Ab alone. (D) The effect of increasing the [Ca²⁺]_e to 1.2 mmol/L or chelating extracellular Ca²⁺ with EGTA (10 mmol/L) on the subsequent response to ₂-subunit cross-linking was determined. [Ca²⁺]_e was altered 2 minutes before the addition of the secondary Ab. (E) After coating with primary antibody, the effect of pretreatment of cells with PP1 (10 μmol/L for 3 minutes) on subsequent ₂-subunit cross-linking was determined, and in (F) ₂-subunit cross-linking was performed in fyn-deficient megakaryocytes. Unless otherwise indicated, experiments were performed in [Ca²⁺]_e equal to 200 μmol/L. Each trace is representative of 6 to 12 cells from a minimum of three different mice.