Examination of platelet function and cytosolic calcium flux on addition of Bcl-x_L-inhibitory BH3 mimetics. (A) Washed platelets^3,5  (3.0 × 10⁸/mL) in Tyrode's buffer were treated with ABT-737 (1μM) for up to 180 minutes. At the indicated times, aggregation in response to thrombin (0.1 U/mL) was monitored at 37°C in a 4-channel automated platelet analyser (AggRAM, Helena Laboratories).³  Results represent the mean ± SEM (n = 5; where ***P < .001). (B-C) Washed platelets (3.0 × 10⁸/mL) loaded with Oregon-green BAPTA (OG) and Fura-Red (FR)⁹  were resuspended in Hepes-buffered saline (HBS) and either treated with thrombin (0.5 U/mL), ABT-737 (10μM) or an equivalent volume of DMSO. Calcium flux was monitored continuously, both before and after addition of agonist or drug (indicated by the arrow), for a total of 6-12 minutes, at 535 and 660 nm, using a fluorescence plate reader and Wallac 1420 workstation software (Wallac VICTOR²  1420 multilabel counter, Perkin Elmer). Traces represent data collected from 1 experiment representative of at least 4 independent experiments. Note: for all experiments, similar results were obtained on addition of ABT-263 or ABT-737 (1-10μM). Moreover, similar results were obtained under a variety of different assay conditions (washed platelets resuspended in Tyrode buffer, in the presence or absence of EGTA) or using different calcium indicator dyes (Fluo-3).