| Assay Method Information | |
| | Glutamine Uptake |
| Description: | The glutamine uptake assays in HEK293 cells were carried out in 96 well plates (CulturPlate-96, Perkin Elmer). Cells were plated at a density of 35,000 cells per well 24 hours prior to carrying out the assay. Each set of conditions is carried out in triplicate. The wells are washed three times with 100 uL of assay buffer at pH 6.0 (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). 1H-glutamine (500 nM) in the same buffer is added along with inhibitor and allowed to incubate for 15 min at 37° C. Following incubation, the H-glutamine/inhibitor is removed and the cells are washed three times with buffer. The cells are then lysed by the addition of 50 uL 1M NaOH. 150 uL of scintillation fluid (Microscint 40, Perkin Elmer) is added and the plates are counted on a scintillation counter (Topcount, Perkin Elmer). |
| Affinity data for this assay | |
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