Glucose free rpmi

Sorted B cells were cultured in vitro under normoxic or hypoxic conditions and stimulated as described herein. For the last 30 minutes of culture, the cell culture medium was replaced with glucose-free RPMI (Thermo Fisher Scientific) supplemented with 1000 U/mL pen/strep (MilliporeSigma) and 50 μM of 2-NBDG (Invitrogen, Thermo Fisher Scientific). Prior to addition of the glucose-free cell culture medium, the media were left to equilibrate under the respective normoxic or hypoxic conditions of the cells. Following incubation, cells were washed in PBS, incubated with viability dye followed by Fc blocking step and extracellular staining as described herein, and then immediately acquired on a 4-laser Fortessa analyzer (BD). Relative mitochondrial mass was estimated based on incorporation of MitoTracker Green (Thermo Fisher Scientific). RA patient–derived B cells were isolated and stimulated under normoxic and hypoxic conditions as described herein. During the last 30 minutes of culture, cells were washed and resuspended in RPMI without FBS supplemented with 20 nM of MitoTracker Green. Cells were washed and stained for viability and expression of PD-1 before acquisition on a 4-laser Fortessa analyzer.