CHEMBL5729962

Uptake Assay: A cDNA clone expressing human SGLT1/SGLT2 was bought from GenerScript. Having the sequence information, it was built into pcDNA5 carrier by using traditional molecular biology methods, and then the expression plasmids were transfected into Flp-in CHO cells by using Lipofetamin 200 liposomal transfection method. The transfected cells were screened for hygromycin resistance, and the single-cell clone was screened out through the process of gradient dilution. Having obtained the single-cell clone, the uptake assay of 14C-AMG in FLP-in CHO cells stably expressing SGLT1/SGLT2 was evaluated.Cells were seeded at a density of 3x104 cells per well, uptake assay was carried out after adherent cells were cultured overnight. At least 12 hours later of culture, cells were washed once by 150 microliters per well of the absorption solution KRH-NMG (120 mM NMG, 4.7 mM KCl, 1.2 mM MgCl2, 2.2 mM CaCl2, 10 mM HEPES, pH 7.4 with HCl). To every well that was cleaned with buffer KRH-Na+ and KRH-NMG.