Vivaspin 20 50 kda mwco

Anti-PD-1 mAb clone G4 was grown from the G4 hybridoma cell line. Hybridoma cells were grown in hybridoma serum free media supplemented with L-glutamine. After one week, the supernatant was harvested and run over a HiTrap protein G column (GE Healthcare, Little Chalfont, Buckinghamshire, UK), then eluted according to the manufacturer's protocol. G4 mAb was concentrated by membrane ultrafiltration with a Vivaspin 20 50 kDa MWCO (GE Healthcare) and concentration was measured by Nanodrop ND-1000 Spectrophotometer.

Dimeric peptide-loaded MHC-Ig was produced as previously described[11 ]. Briefly, Kb-Ig was produced using hybridoma cell lines in serum free media and captured on a NP sepharose column. Kb-Ig was loaded with the SIYRYYGL peptide (GenScript, Piscataway, NJ) using active protein folding via buffer exchange and washed using membrane ultrafiltration with a Vivaspin 20 50 kDa MWCO (GE Healthcare). Non-cognate TRP2 peptide (SVYDFFVWL), (GenScript), was loaded in the same way. Fluorescent KbSIY was produced by labeling with Fluorescein-5-Isothiocyanate (FITC 'Isomer I') (Sigma Aldrich, St. Louis, MO) per manufacturer’s recommendations. Briefly, a 1 M carbonate-bicarbonate buffer at a pH of 9.0 was added at a 1:10 ratio to the KbSIY. FITC-isothiocyanate was dissolved in DMSO (Sigma Aldrich) at a concentration of 1 mg/mL and added to the KbSIY at a 5:1 molar ratio and allowed to react for 2 hours at room temperature. FITC-KbSIY was washed using membrane ultrafiltration at a 50 kDa MWCO (GE Healthcare). To make staining MHC-Ig, loaded dimeric MHC-Ig was biotinylated by reacting a 20-molar excess of EZ-Link^™ Sulfo-NHS-Biotin (ThermoFisher) for 30 minutes at room temperature and then washing the protein using membrane ultrafiltration.