| Assay Method Information | |
| | 3H-Amino Acid Uptake Assays |
| Description: | Live-cell amino acid uptake assays using HEK293 cells were carried out in 96-well plates (CulturPlate-96, Perkin Elmer). 96-well plates were coated with poly-D-lysine prior to the assay. Cells were plated at a density of 35,000 cells per well 24 h prior to carrying out the assay. Each set of conditions was replicated at least three times, technically and biologically. Cells were washed three times with 100 μL of assay buffer (containing 137 mM NaCl, 5.1 mM KCl, 0.77 mM KH2PO4, 0.71 mM MgSO4.7H2O, 1.1 mM CaCl2, 10 mM D-glucose, and 10 mM HEPES) to remove cell media. 3H-amino acid (500 nM) in the same buffer was added concomitantly with V-9302 and allowed to incubate for 15 min at 37° C. For ASCT2-mediated 3H-glutamine uptake assays, 5 mM of the system-L inhibitor 2-amino-2-norbornanecarboxylic acid (BCH) was added and the assay buffer was adjusted to pH 6.0. For selectivity studies, no BCH was added and the assay was conducted at pH 7.4. Following the incubation period, the 3H-glutamine/inhibitor was removed and the cells were washed three times with assay buffer. The cells were then lysed by the addition of 50 μL of 1 M NaOH. For reading, 150 μL of scintillation fluid (Microscint 40, Perkin Elmer) was added and the plates were counted on a scintillation counter (Topcount, Perkin Elmer). Fifty percent inhibitory concentrations (IC50) were calculated using GraphPad Prism version 6 for Mac OS X, GraphPad Software, San Diego Calif. USA, www.graphpad.com. |
| Affinity data for this assay | |
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