2 nbd glucose

The fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBD-glucose; Invitrogen, Darmstadt, Germany) was used to measure the relative uptake of glucose by flow cytometry. In each condition, cells were incubated with 2-NBD-glucose (30 μM) for 1 hour at 37 °C, subsequently washed twice in cold PBS and subjected to flow cytometry (BD Biosciences, Heidelberg, Germany) in fluorescence channel FL1. Data were analyzed using the FlowJo Software.

The fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBD-glucose; Invitrogen, Darmstadt, Germany) was used to measure the relative uptake of glucose by flow cytometry. In each condition, cells were incubated with 2-NBD-glucose (30 μM) for 1 hour at 37 °C, subsequently washed twice in cold PBS and analyzed by flow cytometry (BD Biosciences, Heidelberg, Germany) in fluorescence channel FL1. To determine the effect of SGLT1 inhibition on glucose uptake, cells were incubated with 1 mM of the SGLT1 inhibitor, Phlorizin (1 mM; #274313; Sigma). To determine the effect of Na⁺ on total glucose uptake by SGLT1 inhibition, cells were incubated for 2 h with either HEPES or Na⁺-free Hepes. The solutions were composed of (in mM): standard Hepes: 115 NaCl, 5 KCl, 1 CaCl₂, 1.2 MgSO₄, 2 NaH₂PO₄ 10 glucose, 32.2 Hepes or sodium free Hepes: 132.8 NMDG Cl, 3 KCl, 1 CaCl₂, 1.2 MgSO₄, 2 KH₂PO₄, 32.2 Hepes, 10 mannitol, 10 glucose. All chemicals were purchased from Sigma.