Glucose uptake glo assay

Glucose uptake by Th17 cells was determined using the Glucose Uptake-Glo Assay (Promega), as per the manufacturer’s instructions. Cells were seeded at 2 × 10⁵/well and exposed to 20 μM UCB for the last 6 h of culture, as indicated above. After washing with 1xPBS, 1 mM 2-deoxyglucose was added to the cultures. After incubation for 10 min at room temperature, Stop and Neutralization Buffers were added, followed by 2-deoxyglucose-6-phosphate Detection Reagent. Luminescence was recorded after 3 h incubation at room temperature using an Agilent BioTek Synergy 2 multi-mode microplate reader (Agilent).

For measuring 2DG uptake, Glucose Uptake-Glo Assay (Promega, J1341) was performed as manufacturer’s instruction. Briefly, cells were rinsed with glucose-free IMDM twice, then 50 μL of 1 × 10⁵ K562 cells was seeded per well of a white 96-well plate. Cells were then incubated with 1 mM 2DG at 25 °C for 1 hour, and followed by subsequently adding stop buffer, neutralization buffer, and detection buffer. Luminescence was measured with a plate reader (TECAN, Infinite® 200 PRO). For measuring 2NBDG uptake, K562 cells were pre-washed with glucose-free IMDM, incubated with 300 μM 2NBDG at 37 °C for 30 mins^{14 (link)}, then washed with GM twice, and subjected to flow cytometric analysis. To examine the effect of GLUT1 inhibitors on 2DG/2NBDG uptake, cells were pre-treated with GLUT1 inhibitor as described above, prior to the 2DG/2NBDG incubation. For 2DG uptake assay on purified splenic T and B cells, cells were first isolated with EasySep^TM Mouse T cell Isolation kit (StemCell^TM, 19851) and EasySep^TM Mouse B cell Isolation kit (StemCell^TM, 19854) as manufacturer’s instruction respectively, followed by Glucose Uptake-Glo Assay.