Mwreg30

All animal experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986 and were approved by the UCB Animal Welfare and Ethical Review Body. Male BALB/c mice greater than 6 weeks of age (Charles River, UK) were used.
Acute ITP: Mice were dosed with either 10 mg/kg Fc hexamer, 1000 mg/kg IVIG or PBS control i.v. One hour later platelet loss was induced by the intraperitoneal administration of 1 µg/mouse anti-CD41 (MWReg30; EBioscience). Blood samples were taken immediately prior to Fc hexamer/IVIG dosing and 24 h post anti-CD41 administration to measure baseline and final platelet numbers. Statistical analysis was carried out by one-way ANOVA and Dunnetts multiple comparison test.
Chronic ITP: Platelet loss was induced by continuous subcutaneous infusion of anti-CD41. Mice were implanted with an Alzet subcutaneous osmotic minipump discharging anti-CD41 at 82.5 µg/ml at a flow rate of 0.5 µl/h. Blood samples were taken prior to minipump insertion and again 72, 96, 120, 144, and 168 h later and used to determine platelet number. IVIG was administered as a single dose of 1000 mg/kg, i.v. at 72 h post implantation of the minipump. Fc hexamers were dosed daily, at 10 mg/kg i.v., from 72 h post implantation. Control animals received PBS. Statistical analysis was carried out by one-way ANOVA and Dunnetts multiple comparison test.

Murine blood samples were obtained by retroorbital puncture using citrated capillary. Whole blood samples were centrifuged at 1,300 g for 5 min and resuspended in 1 mL of Tyrode's buffer (134 mM NaCl; 20 mM HEPES; 12 mM NaHCO₃; 5 mM glucose; 2.9 mM KCl; 1 mM MgCl₂; 0.34 mM Na₂HPO₄; pH 7.4). Due to the limited volume of blood samples obtained, flow cytometry was used to test platelets activation and aggregation as previously reported (32 (link)). Aliquots of diluted blood (30 μL) were incubated for 15 min at room temperature with the appropriate anti-mouse monoclonal antibody: APC-conjugated rat anti-integrin alpha IIb (GPIIb, CD41) (6.7 μg/mL; clone MWReg30; eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) and PE-labeled rat anti-mouse integrin αIIbβ3 (GPIIbIIIa, CD41/61) (25 μg/mL; clone JON/A; Emfret Analytics, Würzburg, Germany) were used to detect platelets activation. PE-labeled CD9 and FITC-labeled CD9 (5 μg/mL; clone MZ3; BioLegend, San Diego, CA, USA) were used to visualize aggregation and the double positive population was considered the aggregated platelets. Thrombin (1 U/mL) was added when necessary. To remove excess antibody, aliquots were centrifuged (2,250 g for 5 min) and resuspended in 150 μL of Tyrode's buffer. Analysis was immediately performed in a BD Accury™ C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

For cell sorting and analysis, monoclonal antibodies to CD41 (MWReg30, eBioscience), CXCR4 (2B11, eBioscience), CD11b (M1/70, eBioscience), F4/80 (BM8, eBioscience), Gr-1 (RB6-8C5, Biolegend), Ly6C (HK1.4, Biolegend), CD11c (N418, eBioscience), CD45.1 (A20, eBioscience), CD45.2 (104, Biolegend), CD4 (GK1.5, eBioscience), CD8 (53–6.7, Biolegend), INF-γ (XMG1.2, Biolegend), IL4 (11B11, Biolegend), CD34 (RAM34, eBioscience), Sca-1 (D7, Biolegend), c-kit (2B8, Biolegend), CD135 (A2F10, Biolegend), CD3ε (145–2 C11, Biolegend), CD45R (RA3-6B2, Biolegend), TER-119 (Ter-119, Biolegend), IgM (II/41, eBioscience), FγRII (93, Biolegend), IL-7R (A7R34, Biolegend), TNFα (MP6-XT22, Invitrogen), IL-6 (MP5-20F3, Biolegend), OVA257-264 (SIINFEKL) peptide bound to H-2K^b (eBio25-D1.16 (25-D1.16), Invitrogen) and IL-2 (JES6-5H4, eBioscience) were used where indicated.