Oxygen-glucose deprivation and reperfusion (OGD/R) procedure

Figure 1B shows a schematic diagram of experimental process. We established an in vitro model of cerebral ischemia by subjecting cells to oxygen-glucose deprivation (OGD). For cells undergoing OGD, cultures were transferred to a multi-gas incubator containing a gas mixture with 1% O₂. The medium was replaced with a pre-warmed (37°C) glucose-free DMEM. For removing dissolved oxygen, the solution was bubbled with an anaerobic gas mixture (95% N₂, 5% CO₂) for 1 hour. When dissolved oxygen was less than 0.1 PPM (mg/L) using a dissolved oxygen measuring kit (CHEMetsR kit, K-7501, CHEMetrics Inc, Midland, VA, USA), cell cultures subjected to OGD were incubated in glucose-free DMEM for 4 hours in a multi-gas incubator and then incubated under normoxic conditions in culture medium containing 10% FBS and glucose for additional 24 hours for reperfusion (OGD/R). We administered 30 ng/mL MIF recombinant (concentrations were selected as per Moon et al., 2012) (Cat# ab7207, Abcam, Cambridge, England, RRID:AB_305760) or 50 μM of the MIF antagonist ISO-1 ((S,R)-3-(4-Hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid, methyl ester, MIF antagonist, Merck, Kenilworth, NJ, USA) after 4 hours of OGD and cultures were incubated under normoxic conditions containing 10% FBS and glucose for additional 24 hours. Cultures in the control group were maintained in high glucose containing DMEM under normoxic conditions for 28 hours. To compare cell viability between different treatments, we assigned cell cultures to one of four groups: control, OGD/R, OGD/R with MIF (MIF), OGD/R with ISO-1 (ISO-1).