Anti complement c3b ic3b apc antibody clone 3e7 c3b

Platelets from healthy volunteers with known HLA typing were isolated from Platelet Rich Plasma (PRP), by centrifuging citrated whole blood at 125 g for 20 minutes, optimized with methods described previously to avoid platelet activation [Citation12, Citation16] . Hereafter, 10 vol% ACD (acid citrate dextrose, 85 mM Na 3 -citrate•2 H 2 O, 71 mM citric acid•H 2 O and 111 mM D-glucose) was added. The PRP was centrifuged (850 g for 8 min) and washed 2 times with wash buffer (WB; 36 mM citric acid•H 2 O, 103 mM NaCl, 5 mM KCl, 5 mM EDTA, 5.6 mM D-glucose, pH 6.5). The platelets were set to a concentration of 6 × 10 8 cells/mL in PBS and incubated for 20 min at RT with 3.75 µM PKH26 (Sigma Aldrich) on a roller bank. 10 vol% FCS was added to terminate the labeling process and the labeled platelets were washed with WB and resuspended in PBS +0.5% BSA. 5 × 10 6 platelets were incubated with equal volumes of recombinant anti-HLA antibodies and pooled complement sufficient human serum, for 30 min at RT. For some conditions, the serum was pre-incubated for 30 min at 56°C to inactivate complement. The platelets were washed 3 times with PBS +0.5% BSA +5 mM EDTA and resuspended in macrophage culture medium. Complement deposition (C3b) was assessed by staining a small fraction of the platelets with anti-complement C3b/iC3b-APC Antibody Clone: 3E7/C3b (1/250, Biolegend)