Quantifying Glucose Uptake and Glutamine

DC were treated as above, and after 17 hours, cells and supernatants were collected alongside blank medium as controls. Extracellular glucose was quantified with a glucometer against glucose standards essential as previously described [1 (link)]. Glucose levels (mg/dl) in the blank medium were determined alongside DC +/- treatments. Glucose uptake was calculated by subtracting the glucose in the control, IAV, IAV^BPL, PolyIC, R848, or LPS medium from the cell-free blank medium. Internal and external glutamine were quantified from supernatant and cell lysates, respectively, following the Glutamine/Glutamate Glo assay manufacturer's protocol (Promega, Madison, WI). Briefly, samples were prepared in PBS, diluted to fit in to the linear range of the glutamine or glutamate standard curve, and.25 μl sample or standard transferred to a 96 well luminescence assay plate and reaction allowed to proceed for 30 minutes at room temperature. Next, substrate detection reagent was added and incubated for an additional 60 minutes and metabolite detection was performed using a bioluminescent NADH detection method with luminescence measured using a BMG CLARIOstar microplate reader.