DULBECCO MEM MEDIUM

Protocol tips

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	cells were trypsinized and resuspended (16–32 × 106 cells/mL; >95% viability) in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin (sDMEM) containing 20 mM HEPES.

Protocol tips
cells were trypsinized and resuspended (16–32 × 106 cells/mL; >95% viability) in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin (sDMEM) containing 20 mM HEPES.

Publication protocol

The human glioblastoma T98G and U-87MG cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured as previously described14. On the day of the experiment, the cells were trypsinized and resuspended (16–32 × 106 cells/mL; >95% viability) in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin (sDMEM) containing 20 mM HEPES. For experiments testing the components of the medium (glucose 5.5 mM or 11 mM, glutamine, sodium bicarbonate and FBS), cells were resuspended in the corresponding experimental medium as described in the figure legends. Cell suspensions were maintained at room temperature (ca. 23 °C) and used within 2.5 h.

The data reported here are from experiments conducted over 18 months; as the cells were expanded from different frozen aliquots, and the components of the medium (e.g., FBS) were from different batches, absolute mean values of some variables can be expected to vary (e.g., maxOCR can oscillate up to 25%) more than the standard error of the mean when experiments performed a couple of months apart are compared. Nonetheless, we would emphasize that each experimental protocol was conducted within 2 to 4 weeks and that these variations were not observed; moreover, the effects of treatments were consistent throughout the whole study.

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