Glucose (2-NBDG) uptake assay

Glucose (2-NBDG) uptake assay:

Transfection:

• Seed 1 x 10^⁶ MCF7 cells/ per well in 6 well plate in DMEM with 10% FBS and incubate at 37C for 24 hours in 5% CO₂ incubator.
• MCF7 cells were transfected with SMAR1 siRNA and control siRNA by lipofectamine RNAiMax^TM (Ambion) according to the manufacturer’s protocol. Amount of siRNA and lipofectamine RNAiMax^TM are used as mentioned in the table:
  1. ul

2-NBDG treatment followed by FACS analysis:

• 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. 
• Cells were harvested by trypsinization followed by washing with ice cold 1x PBS. (Repeat the washing twice).
• Cells were divided in two tubes and centrifuged at 2000 RPM for 3 mins at 4C. 
• One tube was used for western blot to confirm the knockdown.
• Remaining tube was further resuspended in 500 ul of ice cold 1x PBS. 
• 1 μg/ml propidium iodide (PI) was added to distinguish the viable cell population before the FACS analysis. 
• 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.