Glucose uptake assay

One × 10⁴ HCT116 cells from control-scrambled, FFAR2 KD and FFAR 2/3 double KD clones were plated in each well of a 96 well plate. Next day, cells were washed twice with phosphate buffer saline and incubated in glucose free DMEM. Glucose uptake was determined using Glucose uptake assay kit (Cayman Chemicals, MI, United States) following manufacturers protocol. Briefly, cells were incubated with 100 μg/ml fluorescent 2-N-7-Nitrobenz-2-oxa-1, 3-diazol-4-yl-Amino-2-Deoxyglucose (2-NBDG) in glucose free medium for 1h. Cells were washed with assay buffer 3 times and analysed immediately. 2-NBDG taken up by cells was detected on a Tecan Infinite 200 pro fluorimeter (Tecan Group Ltd. Männedorf, Switzerland) with fluorescent filters designed to detect fluorescein (excitation/emission = 485/535 nm). Cells without 2-NBDG incubation were used as blank control.