| Assay Method Information | |
| | SGLT2 Glucose Transport Test |
| Description: | 1) Experimental buffer: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1.2 mM magnesium chloride (MgCl2), 4.7 mM potassium chloride (KCl), 2.2 mM calcium chloride (CaCl2)) and 120 mM sodium chloride (NaCl).2) Termination buffer: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1.2 mM magnesium chloride (MgCl2), 4.7 mM potassium chloride (KCl), 2.2 mM calcium chloride (CaCl2), 120 mM sodium chloride (NaCl) and 1 μM LX4211.3) The compound was diluted with 100% dimethyl sulfoxide (DMSO) with a starting concentration of 10 μM and 8 points of 5-fold serial dilution.4) 6 μM [14C]-labeled methyl α-D-glucopyranoside was prepared with experimental buffer.5) The cells were treated with 49 μL of experimental buffer, 1 μL of gradient diluted compound, and 50 μL of 6 μM [14C]isotope-labeled sugar solution at 37° C. for 2 hours.6) The liquid in the well was aspirated and the cells were rinsed 3 times with termination buffer.7) The cells were lysed with 50 μL of 10% sodium hydroxide solution, the cell lysate was aspirated into the scintillation tube, and 2 mL of scintillation fluid was added.8) An isotope detector (Tricarb) was used to read.9) The data was calculated by GraphPad Prism 5.0 software: log(inhibitor) vs. response—Variable slope to obtain the IC50 value of the test compound. |
| Affinity data for this assay | |
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