Fam lehd fmk

The surface exposure of phosphatidylserine (PS) in washed platelets was assessed by measuring the binding of (FITC)-labeled Annexin V (BD Pharmingen, San Diego, CA, USA). Washed platelets were resuspended in annexinV binding buffer (10 mM of HEPES, 10 mM of NaOH, 140 mM of NaCl, 2.5 mM of CaCl₂, pH 7.4) and labeled with FITC-annexinV. After incubation for 15 min at RT in the dark, the samples were analyzed by flow cytometry.
To analyze the caspase-3, -7, -8, and -9 activity, PRP was diluted 10-fold with isotonic HEPES buffer containing 2 mM of CaCl₂ and 2 mM of Gly-Pro-Arg-Pro acetate (Sigma Aldrich, Madrid, Spain) to prevent fibrin formation, and either FAM-DEVD-FMK, FAM-LETD-FMK, or FAM-LEHD-FMK (Millipore, Madrid, Spain). The samples were analyzed by flow cytometry.

We assessed the surface exposure of phosphatidylserine in washed platelets by measuring the binding of FITC-labelled annexin V (BD Pharmingen, Madrid, Spain). Briefly, washed platelets were resuspended in annexin V binding buffer (10 mM HEPES, 10 mM sodium hydroxide, 140 mM sodium chloride, 2.5 mM calcium chloride, pH 7.4) and labelled with FITC-annexin V. After a 15-min incubation period with either buffer or 1 µM ionomycin (Sigma, Madrid, Spain) at room temperature in the dark, the samples were analysed by flow cytometry.
To analyse active caspase-3, -8 or -9, we diluted PRP 10-fold with isotonic HEPES buffered saline with calcium ion (150 mM sodium chloride, 2 mM calcium chloride, 2 mM magnesium chloride, 2 mM HEPES, pH 7.4), 2 mM Gly-Pro-Arg-Pro (SIGMA, Madrid, Spain) and either FAM-DEVD-FMK, FAM-LETD-FMK or FAM-LEHD-FMK (Millipore, Madrid, Spain).