| Assay Method Information | |
| | Assay of the Citric Acid Cycle Activation |
| Description: | NaCT-CHO and pME-CHO were plated at 20,000 cells/well into white CulturPlate -96 (PerkinElmer) two days before the assay. Prior to assay incubation, the cultured plates were washed twice with washing buffer, 10 mM HEPES-Tris(pH7.4) containing 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2. The compounds to be tested were dissolved and diluted in DMSO (Wako Pure Chemical industries) to 1,000 times of a final concentration, and further diluted to two times as high as the final concentration in assay buffer, 10 mM HEPES-Tris(pH7.4) containing 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2. The range of final concentrations was properly determined based on the test compounds activity. Each 25 μL compound solution was added to well and subsequently 25 μL radio-labeled substrate solution containing 0.4 mM (0.4 MBq/mL) [1,5-14C]-citric acid (PerkinElmer) in the assay buffer was added. After 1 hour incubation at 37° C., the reaction mixture was discarded and washed three times with pre-chilled washing buffer and then 0.1 mL MicroScint 20 (PerkinElmer) was added to well. The plate was sealed with TopSeal-A (PerkinElmer) and the radioactivity was measured using a TopCount (PerkinElmer). Non-specific activity (NS cpm) and total radio activity (Total cpm) were determined by counting of pME-CHO plated wells and NaCT-CHO plated wells without compounds, respectively. Diffusion of [14C] CO2 was able to be estimated from residual radioactivity (R cpm) by an equation (Total−R)/(Total−NS)×100(%). The difference of Total and R was disappeared in the presence of 0.1 μM antimycin A. EC50 values were calculated by regression analysis using SAS Statistical Analysis System (SAS institute Japan Ltd. Release 9.1). |
| Affinity data for this assay | |
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