Radiolabeled Amino Acid Uptake Assay to Quantify Cellular Uptake of Amino Acids

Amino acid transporters are membrane-bound proteins that mediate amino acid transfer across cell membranes, regulating vital cellular processes.

To study amino acid uptake in vitro, take a culture of mouse bone marrow-derived stromal cells. Aspirate the growth medium. Add a pre-warmed buffer supplemented with glutamine — an amino acid — labeled with radioactive hydrogen. Incubate for the required duration.

The pre-warmed buffer provides physiological temperature and an appropriate pH required for the cellular uptake of glutamine. Glutamine transporters consist of transport domains — responsible for substrate translocation via sodium ion-coupled co-transport — and membrane-anchored scaffold domains.

The transport domain binds to radiolabeled glutamine along with sodium ions from the buffer. Upon binding, it releases the molecules into the cytoplasm. Simultaneously, the transport domain translocates intracellular sodium ions and neutral amino acids outside the cell, resulting in cellular homeostasis.

Wash the cells with ice-cold buffer, terminating the reaction and removing any extracellular radiolabeled glutamine. Add a detergent solution. The detergent lyses the cells, causing the release of intracellular radiolabeled glutamine.

Centrifuge the lysate. Add the glutamine-containing supernatant into a vial with a scintillation cocktail, and place the vial inside a scintillation counter.

The radiolabeled glutamine molecules emit high-energy beta particles. The scintillators in the cocktail absorb the particles and emit light pulses that are detected by the scintillation counter.

Measure the radioactivity to estimate the cellular glutamine uptake.

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.