Glucose uptake assay

GS Georg Schneditz
JE Joshua E. Elias
EP Ester Pagano
MC M. Zaeem Cader
SS Svetlana Saveljeva
KL Kathleen Long
SM Subhankar Mukhopadhyay
MA Maryam Arasteh
TL Trevor D. Lawley
GD Gordon Dougan
AB Andrew Bassett
TK Tom H. Karlsen
AK Arthur Kaser
NK Nicole C. Kaneider
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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.

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