| Assay Method Information | |
| | Sodium-Dependent Glucose Transporter (SGLT) Assay |
| Description: | The SGLT activity is measured as sodium-dependent 14C-AMG uptake in the above cell lines described as follows. One hundred uL of culture medium containing 30,000 cells are seeded to each well of a 96-well BioCoat poly-D-lysine plate (Becton Dickson) and cultured at 37° C. overnight. The culture medium is aspirated and cells are washed twice with 200 uL of Reaction Buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, MgCl2, and 14 mM N-2-hydroethylpiperrazine-N'-2-ethanesulfonic acid (Hepes), pH 7.5). The excess buffer is tapped out onto paper towels. Thirty-five uL of Reaction Buffer are added to each well. Five uL of a 10% dimethylsulfoxide (DMSO) in Reaction Buffer containing varying concentrations of test compound or no compound as a control, is dispensed into the each well. The reaction is initiated by adding 10 uL of 14C-AMG in Reaction Buffer to make a final concentration of 4 uM. The plate is incubated at 37° C. for 125 minutes. The reaction is terminated by aspirating off Reaction Buffer and then washed three times with 200 uL of ice cold Reaction Buffer. Manual aspiration is applied to ensure the complete removal of Reaction Buffer. Ten uL of 0.1 N NaOH is added to each well and then 100 uL of Supermix scintillation cocktail (PerkinElmer) is added. After mixing, the scintillation signal in the plate is counted in a MicroBeta (PerkinElmer). A ten-dose response curve is fitted to an empirical four-parameter model using ActivityBase (ID Business Solution) to determine the inhibitor concentration at half-maximal inhibition (IC50). |
| Affinity data for this assay | |
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