Cd62p bv421

Citrate-anticoagulated whole blood was centrifuged at 300 × g for 10 min to gain PRP. 100 µl of PRP were incubated for 6 min at 37 °C. After another 6 min of stimulation with ADP (5 µM) at 37 °C, the following antibodies were added and incubated for 30 min at 37 °C: CD41/61-APC-Cy7 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD42b-Pe-Cy7 (Invitrogen, Massachusetts, USA) and CD62P-BV421 (BD Biosciences, Franklin Lakes, USA). Before measurement with BD FacsVerse® (BD Biosciences, Franklin Lakes, USA), the solution was diluted 1:100 in phosphate-buffered saline (PBS). Data were processed and analyzed using Flow-Jo® (BD Biosciences, Franklin Lakes, USA)

After blood collection and PRP separation, samples were immediately processed for flow cytometry (FC). PRP (20 μL of 1 × 10⁸ cells/mL solution) was mixed with the antibodies cocktail (20 μL in PBS, phosphate-buffered saline) and the test stimulus (40 μL in PBS). As stimuli, we used buffer only (negative control), 10 μM ADP (positive control for platelet activation) or L. interrogans Copenhageni (2 × 10⁶ cells/mL). The reactions were incubated for 5, 10 or 30 min at RT, protected from light, then stopped and fixed by the addition of 500 μL 0.2% formaldehyde. The following conjugated antibodies dilutions were used: 5 μL CD41a-APC, 3 μL CD62P-BV421, 5 μL PAC-1-FITC, 5 μL CD63-FITC – all from BD Bioscience. Flow cytometric acquisition was performed with a FACS Canto II after optimization of settings using the cytometer setup and tracking beads. Instrument and reagents were from BD Bioscience. Data were analyzed using FlowJo version 7.6.5. The experiments were performed with four different blood donors, at least in duplicates. Data from 20,000 events in the CD41a-positive gate were collected for each sample.