Encyclopedia of Experiments: Biology
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Transcript
To begin amino acid uptake assay, plate 5 x 104 ST2 cells, in each well of a 12-well tissue culture plate in alpha-MEM medium supplemented with FBS, penicillin, and streptomycin. Plate extra wells of cells to quantify the cell number per condition for the normalizations.
Next, incubate cells in a humidified cell culture incubator at 37 degrees Celsius with 5% carbon dioxide for two to three days until confluent. After incubation, aspirate the medium, and wash the cells twice with one milliliter of PBS at pH 7.4. Then, aspirate the PBS before washing the cells once, with one milliliter Krebs Ringers HEPES, or KRH.
Incubate cells with 0.5 milliliters of freshly-prepared KRH containing four microcuries per milliliter L-[3,4-3H]-Glutamine working media for five minutes. Post-incubation, collect the radioactive medium to dispense into the liquid waste container. Then, wash the cells three times with ice-cold KRH to terminate the reaction.
Next, add one milliliter of 1% sodium dodecyl sulfate to each well, and triturate the mixture 10 times to lyse and homogenize the cells. Then, transfer the cell lysates to a 1.5-milliliter tube, and clarify the lysate by centrifuging at a minimum speed of 10,000 g for 10 minutes.
Transfer the supernatant to a scintillation vial, containing eight milliliters of the scintillation solution, and read radioactivity in counts per minute using a scintillation counter.
From the remaining non-radioactive plates of cells, trypsinize, resuspend, and count the cells to estimate the number of cells in the lysed radioactive cultures. Using a hemocytometer, count the number of cells per non-radioactive well for each experimental condition.
