2-DG uptake assays were performed as previously described49 (link). Primary mouse or human keratinocytes were seeded in triplicate in 12-well plates overnight (50000), washed twice with PBS (Sigma, D8537), incubated in basic KSFM (without any supplementation) or serum free DMEM/F12 medium (Thermo, 11320033, for SCCT8 cells) for 2 hours, washed twice with KRHP buffer, and incubated in 0.45 ml KRHP buffer to each well, and starved for 30 minutes. For inhibition of glucose transporter, Cytochalasin B (10 µM, Sigma, C6762) and phloretin (100 µM, Sigma, P7912) were added to the KRHP medium for another 15 minutes. Uptake was initiated by adding 1µCi 3H 2-DG (25–30 Ci/mmol, PerkinElmer, NET549) and 0.1 mM unlabeled 2-DG (Sigma, D8375) in KRHP to each well for 10 or 20 min. Transport activity was terminated by the rapid removal of the uptake medium and washing 3 times with cold PBS with 25 mM glucose (Sigma, G7528). Cells were lysed with 0.5 ml of 0.5 M NaOH (Fisher Scientific, SS255-1) and neutralized with 0.5 ml or 0.5 M HCl (Sigma, 320331), mixed well. 250 µl of the lysate was transferred to a scintillation vial with scintillation solution, and the sample was quantitated by liquid scintillation counting. Protein concentrations were determined by BCA assay (Thermo, 23227).
P7912
Manufactured by Merck Group
P7912 is a laboratory equipment product manufactured by Merck Group. It is a multi-purpose device designed for scientific and research applications. The core function of P7912 is to provide a controlled environment for various experimental procedures and analyses.
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2 protocols using p7912
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2-DG Uptake Assay in Keratinocytes
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2-DG Uptake Assay in Keratinocytes
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2-DG uptake assays were performed as previously described49 (link). Primary mouse or human keratinocytes were seeded in triplicate in 12-well plates overnight (50000), washed twice with PBS (Sigma, D8537), incubated in basic KSFM (without any supplementation) or serum free DMEM/F12 medium (Thermo, 11320033, for SCCT8 cells) for 2 hours, washed twice with KRHP buffer, and incubated in 0.45 ml KRHP buffer to each well, and starved for 30 minutes. For inhibition of glucose transporter, Cytochalasin B (10 µM, Sigma, C6762) and phloretin (100 µM, Sigma, P7912) were added to the KRHP medium for another 15 minutes. Uptake was initiated by adding 1µCi 3H 2-DG (25–30 Ci/mmol, PerkinElmer, NET549) and 0.1 mM unlabeled 2-DG (Sigma, D8375) in KRHP to each well for 10 or 20 min. Transport activity was terminated by the rapid removal of the uptake medium and washing 3 times with cold PBS with 25 mM glucose (Sigma, G7528). Cells were lysed with 0.5 ml of 0.5 M NaOH (Fisher Scientific, SS255-1) and neutralized with 0.5 ml or 0.5 M HCl (Sigma, 320331), mixed well. 250 µl of the lysate was transferred to a scintillation vial with scintillation solution, and the sample was quantitated by liquid scintillation counting. Protein concentrations were determined by BCA assay (Thermo, 23227).
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