MPA were identified in citrate anticoagulated blood as previously described [8 (link)]. Briefly, whole blood was fixed with 1% formaldehyde (Sigma) 15 min after blood collection. Fixed whole blood was stained with 5 μL CD61-FITC (Dako) to identify platelets and 5 μL CD14-APC (BD Biosciences) to identify monocytes. After lysis of red blood cells, monocytes were collected based on side-scatter properties and positive staining for CD14 using an Accuri C6 flow cytometer (BD Biosciences). MPA were identified as having a positive stain for CD14 and CD61, and were recorded as a percent of 2000 total monocytes collected.
Platelet activation was determined by platelet surface expression of P-selectin, and CD40 with whole blood flow cytometry, as previously described [8 (link)]. Briefly, P-selectin expression was determined with a FITC-conjugated anti-CD62P antibody (BD Biosciences), and CD40 expression was determined with a FITC-conjugated anti-CD40 antibody (BD Biosciences). Gates were established to include platelets with and without aggregation to monocytes. Monocytes were identified by staining with CD14-APC (BD Biosciences), and platelets were identified by staining with CD42-PE (BD Biosciences). The expression of platelet activation markers were assessed individually
Platelet activation was determined by platelet surface expression of P-selectin, and CD40 with whole blood flow cytometry, as previously described [8 (link)]. Briefly, P-selectin expression was determined with a FITC-conjugated anti-CD62P antibody (BD Biosciences), and CD40 expression was determined with a FITC-conjugated anti-CD40 antibody (BD Biosciences). Gates were established to include platelets with and without aggregation to monocytes. Monocytes were identified by staining with CD14-APC (BD Biosciences), and platelets were identified by staining with CD42-PE (BD Biosciences). The expression of platelet activation markers were assessed individually