Pe rat anti mouse cd41

Following dilution of 20 μl of blood with 30 μl acid-citrate-dextrose (ACD) buffer (85 mM trisodium citrate, 65 mM citric acid, 100 mM glucose, pH 5.0) and 50 μl PBS, 1 μl PE Rat Anti-Mouse CD41 (BD Pharmingen, #561850) antibody was added to stain the platelets, and incubated at RT for 30 min. To this, 900 μl of fixative (0.2% formyl saline) was added and incubated for a further 10 min at RT. Finally, 10 μl of this fixed cell suspension was then added to 990 μl PBS. A hemocytometer was used to count whole blood cells using transmitted light, and platelets counted using blue epifluorescence. Four animals at the age of four months and of mixed sex were used for each genotype group, experimental replicates were biological.

Isoflurane was from Schering-Plough Animal Health (Kenilworth, NJ). PE Rat Anti-Mouse CD41, FITC RAT Anti-Mouse CD62P and FITC RAT Anti-Mouse CD45 was from BD Biosciences (Franklin Lakes, NJ). Thrombin was from Parke Davis (Morris Plains, NJ) and Type I Collagen (Vitrogen 100) from Angiotech BioMaterials (Palo Alto, CA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).

The following reagents were used in this study: wild-type smooth strain Escherichia coli (E. coli) (O55:B5, ATCC® 12014™), LPS from E. coli (0111:B4) and RIPA buffer with protease inhibitor cocktail were from Sigma-Aldrich, St Louis, Missouri), pyrogen-free saline (Thermo Fisher Scientific, North Ryde, New South Wales, Australia), Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences, North Ryde, New South Wales, Australia), bovine serum albumin (BSA) (Sigma-Aldrich), alkaline phosphatase (AP) conjugated anti-mouse (Sigma-Aldrich), anti-rabbit and anti-human IgG (Sigma-Aldrich), β2GPI (Hematologic Technologies, Essex Junction, Vermont), isotype control rabbit polyclonal IgG (BD Biosciences) and red blood cell lysis buffer (eBioscience, San Diego, California). Triple-Pure High Impact Zirconium 1.5 mm beads (Benchmark Scientific, NJ) Affinity purified murine IgG2, anti-β2GPI monoclonal antibody and affinity purified polyclonal rabbit anti-β2GPI antibody were produced as previously described^{22 (link), 23} .
PE-rat anti-mouse CD41, APC-Cy7 rat anti-mouse CD45, BV510 hamster anti-mouse CD3e, PerCP-Cy5.5 rat anti-mouse CD4, PE-Cy7 rat anti-mouse CD8a, APC rat anti-mouse CD19, V450 rat anti-mouse LY-6G and LY-6C, PE rat anti-mouse CD11b and mouse Fc block^TM were purchased from BD Biosciences and used at 0.2 mg/mL.