Release of annexin V-binding microparticles from HUVEC after stimulation with different agonistsA) Short-time stimulation (30 min): confluent HUVEC at 48 hrs in culture were incubated with buffer HBSS/BSA only (HBSS), 0.5 % DMSO in HBSS/BSA (DMSO), 10 μM A23187 (A23187), PMA 1 μg/mL (PMA), 10 μM thrombin receptor activating peptide (TRAP), thrombin 1 NIH U/mL (THR), tPA 3.5 kU/mL (TPA), or with 1 μM angiotensin II (AGTII). B) Overnight stimulation (24 hrs): confluent HUVEC at 48 hrs in culture were incubated with medium only (MEDIUM), 0.5 % DMSO in medium (DMSO), TNFα 1000 U/mL (TNF), cycloheximide 50 μg/mL (CHX), TNFα 1000 U/mL with cycloheximide 50 μg/mL (TNF/CHX), lipopolysaccharide E. coli 055:B5 10 μg/mL (LPS), 5 μM camptothecin (CPT), 5 μM camptothecin with 50 μM Z-VAD-fluoromethyl ketone (CPT/ZVAD), or with 50 μM Z-VAD-fluoromethyl ketone. After incubation, supernatant from cell culture was harvested and annexin V-FITC-positive MP were counted. Means of 4 independent experiments ± SD are presented, * = p < 0.05 vs. DMSO (ANOVA).
Šimák et al. BMC Cell Biology 2002 3:11 doi:10.1186/1471-2121-3-11