Recombinant MIF and Antagonist Preparation

Recombinant human MIF (rhMIF) was prepared as described elsewhere (46 (link)) (endotoxin content < 0.1 EU/ml). MIF antagonist MIF098 [3-(3-hydroxybenzyl)-5-methylbenzooxazol-2-one] was dissolved in DMSO at a concentration of 149 µM (47 (link)). The neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9) was obtained from ascites after purification using protein A/G spin column and resuspended at 5.15 mg/ml (48 (link), 49 (link)). A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R&D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained.