2 nbdg

Fresh healthy donor human PBMCs were incubated with CellROX (Thermo Fisher Scientific), NAO (Thermo Fisher Scientific), or 2-NBDG (Carbosynth) according to the manufacturer’s specifications. For all three conditions, cells were stained during the efflux incubation for MitoTracker and otherwise prepared for flow cytometry using the protocol detailed above with a few exceptions. 2-NBDG and NAO panels required substitution of MitoTracker Orange (Thermo Fisher Scientific). The fluorochrome-conjugated primary antibody panel was as follows: CD3 (HIT3a, BioLegend), CD19 (SJ25C1, BioLegend), CD27 (M-T271, BD Biosciences), IgD (IA6–2, BioLegend), and CD10 (HI10a, BioLegend). Compensation for the functional dyes required the use of Ramos cells (ATCC), incubated with CellROX, 2-NBDG, or NAO, respectively.

We assessed a potential mechanism by which OR2H1 enhances glucose metabolism using the fluorescent glucose analog 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (Invitrogen N13195). We diluted 2-NBDG in glucose-free R10 at a concentration of 200μM and incubated with H2009-wildtype (ATCC Cat# CRL-5911, RRID:CVCL_1514) and H2009-OR2H1 knockout cells for 30 minutes at 37°C and 5% CO₂. We then measured the signal in the FITC channel via the BD LSRII flow cytometer to evaluate glucose uptake in the wildtype cells compared to the knockout.