| Assay Method Information | |
| | LAT1 Uptake Inhibition Assay |
| Description: | The ability of compounds to interact with LAT1 was measured using a radiolabeled competition uptake assay with [3H]-Gabapentin ([3H]-GP) (Perkin Elmer) in 96-well plates with LN229 cells (ATCC®). Ten (10)×103 (10,000) cells/well were plated in white, clear bottom plates and were allowed to grow for three (3) days. On the fourth day, the cells were washed and then incubated with 50,000 counts per minute (cpm) of [3H]-GP in phosphate buffered saline (PBS) (VWR International) with increasing concentrations of test compounds in at least triplicate for 15 min. At end of the assay time, the incubation solution was removed, and plates were washed three times (3×) with 100 μL of ice-cold PBS buffer. One-hundred fifty (150) L of scintillation fluid (VWR International) was added to each well, and the radioactivity retained within the cells was measured on a 96-well scintillation counter. Background uptake ([3H]-GP uptake in the presence of 10 mM non-radiolabeled gabapentin (GP)) (MedKoo) was subtracted and data were normalized to the DMSO control ([3H]-GP uptake in the absence of any competitor). Data were fitted to the Michaelis-Menten equation using GraphPad Prism (Version 8.2.0). |
| Affinity data for this assay | |
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