The Glucose Uptake-Glo Assay (Promega, USA) was used according to the supplier’s recommendation. Briefly, BMDMs were seeded into white 96-well plates (Nunc, Denmark) at a cell density of 5 × 104 cells per well and polarized as described above. Cells were washed once with PBS and then incubated with PBS containing 1 mM 2-DG for the given time points (10 to 60 min). The reaction was stopped with stop buffer, and immediately after this, pH was neutralized with neutralization buffer. The detection reagent for 2DG6P was added to the wells. After incubation for 1 hour, luminescence (Centro LB 690 Luminometer, Berthold) was measured using 0.3-s integration every 2 min for 15 min. A standard curve of 2DG6P was used to extrapolate the concentration of 2DG6P in the sample.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.