Wheaton celline bioreactor flasks

For all antibody treatments, the schedules are indicated in the figures. All treatments were delivered by intraperitoneal injection. Mouse anti-mouse IFNAR1 (MAR1-5A3) and isotype control (GIR208, mouse anti-human IFNGR-α chain) were purchased from Leinco Technologies Inc. Each mouse was given 500μg per injection. Hamster anti-IL-1R1 antibody (mIL1R-M147) was obtained from Amgen⁴⁵ . Isotype control was Ultra-LEAF Purified Armenian Hamster IgG Isotype Antibody from Biolegend (400940). Each mouse was given 200μg per injection. Armenian hamster anti-IL1Ra antibody was produced in-house using a previously published hybridoma line^{60 (link)}. Cells were grown in Wheaton CELLine Bioreactor Flasks (Fisher Scientific) according to manufacturer instructions. Media in the cell compartment used ultra-low IgG FBS (ThermoFisher 16250078) to minimize bovine IgG contamination during purification. Cell-free supernatant from the cell compartment was purified using protein G resin (GenScript). IgG was eluted using 0.1M acetic acid, then 10% of total volume of 1M Tris pH8 and 0.5M NaCl was added to neutralize. Size exclusion and buffer exchange to PBS was performed using Amicon Ultra-4 Centrifugal Filter Units (EMD Millipore). The final product was filter sterilized and stored at −80°C. For injections antibody stocks were diluted in sterile PBS and each mouse received 500μg per injection.