αigg f ab 2

Whole splenocytes from naïve hemophilia A mice were stained with 2.6 μM Fluo-3 (Invitrogen) and 5.5 μM Fura Red (Invitrogen) for 45 min at 37°C. To identify the B cell subset of the whole splenocytes suspension a PE-Cy7 CD19 antibody was used (BD Pharmingen). B cell calcium flux was then assessed using flow cytometry (SH800S, Sony). Following 5 min of baseline fluorescence reading, αIgG (10 μg/ml, Southern Biotech), αIgG F(ab)₂ (10 μg/ml, Southern Biotech), αIgG F(ab)₂ + BDD FVIII (11.4 μg/ml) or αIgG F(ab)₂ + rFVIIIFc (14.7 μg/ml) were added and data were acquired for a further 7 min. All samples were then treated with ionomycin (1.4 μM) to elicit a maximal response and then finally quenched with EGTA (5 mM). Data was then analyzed using FlowJoX (Tree Star) and the median ratio of Fluo-3 to Fura Red fluorescence was reported as a measure of intracellular calcium flux.

Naïve and FVIII-exposed B cells as well as 413 cells were incubated with BDD FVIII (11.4 μg/ml), rFVIIIFc (14.7 μg/ml), goat anti-mouse IgG F(ab)₂ (αIgG F(ab)₂, 20 μg/ml, Southern Biotech) or whole goat anti-mouse IgG (αIgG, 20 μg/ml, Southern Biotech) for 30 min at 37°C. Cell lysates were then extracted and separated on an SDS PAGE gel, followed by transfer to nitrocellulose membrane (Bio Rad). Membranes were then blotted for phosphorylated SH2-containing inositol phosphatase (pSHIP, Cell Signaling Technology), SHIP (Santa Cruz Biotechnology), phosphorylated ERK (pERK, Cell Signaling Technology), ERK (Cell Signaling Technology) and actin (Abcam). Detection was carried out using horseradish peroxidase—conjugated (HRP) goat anti-rabbit (Dako) and goat anti-mouse (Southern Biotech) Ig followed by development with an enhanced chemiluminescence substrate (PerkinElmer). Densitometry analysis was performed using ImageJ (NIH) and ratios of phosphorylated to total protein were averaged for three different blots. No statistical analysis was carried out for these data due to the qualitative nature of the assay.