Cd63 pe cy7

Platelet activation was quantified by the upregulation of their surface markers, CD63 and CD62P, by flow cytometry. Briefly, after separation from whole blood, the platelet suspension was kept at 25°C for 4 hours, followed by re-calcification and incubation with E. coli for 15 minutes at 37°C. Then, platelets were stained for 30 minutes in the dark at 4°C with an antibody mixture containing platelet CD42a-FITC (catalog no. 348083, BD Biosciences, San Jose, CA), CD63-PE-Cy7 (catalog no. 25-0639-42, Invitrogen, Carlsbad, CA), and CD62P-PE (catalog no. 555524, BD Biosciences). After staining, the solution was centrifuged at 250×g, 4°C for 5 minutes. The supernatant was discarded and the pellet was resuspended and fixed in PBS containing 0.1% paraformaldehyde (PFA) and 0.1% bovine serum albumin (PBSA) and stored at 4°C until analysis with flow cytometer Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific) within 24 hours from sampling. Platelets were gated as CD42a+ population (Supplementary Figure 1) while CD63 and CD62P were used as platelet activation measures. Flow data analysis was performed with FlowJo software version 10 (Ashland, OR).

After infection, the well contents were collected and centrifuged at 1,000 × g for 2 min. The cell pellet was washed twice with flow buffer (2% FBS in PBS) and resuspended in 50 μl of flow buffer for staining. Neutrophils were stained for 25 min with CD63-PE-Cy7 (Invitrogen) on ice. Neutrophils were washed once in flow buffer and resuspended in flow buffer containing propidium iodide (Invitrogen) to exclude dead cells. Neutrophils were acquired using a Millipore Guava 6HT flow cytometer and analyzed with the InCyte EasyCyte v3.1 software.