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Glucose (2-NBDG) uptake assay

AC Arpankumar Choksi
AP Apoorva Parulekar
RP Richa Pant
VS Vibhuti Kumar Shah
RN Ramakrishna Nimma
PF Priyanka Firmal
SS Smriti Singh
GK Gopal C. Kundu
SS Sanjeev Shukla
SC Samit Chattopadhyay
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MCF7 cells were transfected with SMAR1 siRNA and control siRNA by lipofectamine RNAiMaxTM (Ambion) according to the manufacturer’s protocol. After 24 h of transfection, media was removed and replenished with 10% FBS containing DMEM (without glucose and sodium pyruvate) and incubated at 37 °C for 1 h. 10 μM fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) (Invitrogen) was added to culture media and cells were incubated for 1 h at 37 °C. The 2-NBDG uptake reaction was stopped by removing the incubation medium and the cells were washed with ice-cold 1× PBS. 1 μg/ml propidium iodide (PI) was added to distinguish the viable cell population. For each measurement, data from 10,000 single-cell events were collected using FACS Canto II (BD Bioscience). The percentage of 2-NBDG uptake was calculated from mean fluorescence intensity (MFI) compared with the control.

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