| Assay Method Information | |
| | Inhibition Assay |
| Description: | The entire lactate uptake studies for the inhibition of MCT1 were carried out on RBE4 (Rat Brain Endothelial 4) cells. The expression of MCT1 on these cells was confirmed by Western Blotting. The cells were plated approximately 20-24 hours before the experiment, the number of cells being approximately 105 cells per well. The test compounds were dissolved in DMSO and diluted 1000 times using a solution of HEPES buffer (pH 7.43) which consists of 3 uM 14C-Lactate and 2 uM L-Lactate. The cells were washed twice with 500 uL of HEPES buffer and the cells were allowed to equilibrate for 15 minutes at 37° C. The HEPES buffer was removed and 250 uL of the test sample was added in triplicates. This was repeated for all the compounds, including the controls (CHC and DMSO). After 15 minutes, the compounds were removed from the well and 500 uL of ice-cold stop buffer (0.1 mM CHC solution in HEPES buffer) was added. The plate was kept on ice. Now, the HEPES buffer in one triplicate was removed and DMSO solution was added and immediately removed and ice-cold stop buffer was added. This was considered as "Zero". One triplicate was left blank, which was used for protein assay after lysing the cells. The cells were washed twice with ice-cold HEPES buffer and then 250 uL of 0.1M NaOH in 5% Triton-X solution was added and the plate is kept on a shaker for 40 minutes to lyse the cells. 150 uL of the lysed cells was added into 4 mL of the scintillation fluid in a scintillation vial and scintillation count was obtained in disintegrations per minute (dpm). The percent inhibition values were calculated by taking DMSO as minimum. Concentration study (usually 9-12 dilutions) was done to determine the IC50 of each compound. |
| Affinity data for this assay | |
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