Amino acid uptake and metabolism are essential for osteoblast specification, differentiation, and bone formation. This protocol provides a rapid and sensitive method to evaluate amino acid uptake. Our protocol uses radiolabeled amino acids to quantify amino acid uptake.
It is a cheap, sensitive and a fast method which can be easily modified for different conditions. This protocol can easily be modified to evaluate the uptake of other radiolabeled nutrients like glucose and fatty acids in a variety of tissues as well as primary or transform cells. To begin amino acid uptake assay plate five times 10 to the fourth 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-Ringer's HEPES or KRH. Incubate cells with 0.5 milliliters of freshly prepared KRH containing four microcuries per milliliter L34 tritiated 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 radio activity and counts per minute using the 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 hemacytometer, count the number of cells per non-radioactive well for each experimental condition.
To determine amino acid uptake, flush out the marrow from the bone before weighing the bone shaft. To decellularize the bone, boil one humerus in PBS at 100 degrees Celsius for 10 minutes. Equilibrate both live and boiled humeri in one milliliter of KRH for 30 minutes in the cell culture incubator at 37 degrees Celsius.
Next, incubate the experimental and boiled control bones separately in freshly prepared KRH containing four microcurries per milliliter L234 triciated arginine for up to 90 minutes at 37 degrees Celsius. After removing the radioactive medium, terminate the reaction by washing humerus three times with one milliliter of ice cold KRH. Transfer each bone into a 1.5 milliliter tube with 500 microliters of RIPA Buffer.
Then homogenize the humerus and RIPA Buffer by chopping 100 times with scissors, followed by sonication at 35%amplitude and one second pulse for 10 seconds. Clarified the lysate by centrifugation for 10 minutes at 10, 000 times G at room temperature. Post centrifugation, transfer 200 microliters of the supernatant to scintillation vials containing eight milliliters of the scintillation solution.
Read radio activity using the scintillation counter. Kinetic properties of in vitro L34 tritiated glutamine uptake and confluent ST2 cells were evaluated in the presence or absence of sodium methyl amino isobutyric acid or lithium. The removal of sodium led to a 90%reduction in glutamine uptake.
The presence of lithium increased glutamine uptake by 2%compared to the sodium free condition. Whereas methyl amino isobutyric acid did not affect glutamine uptake. The estimation percentage contributions of system A, N, L or ASC and gamma plus L indicated that most glutamine uptake is mediated by systems ASC and gamma plus L.System N is responsible for roughly 2%sodium dependent glutamine uptake.
And system A showed 0%contribution and uptake. X-VIVO L34 tritiated arginine uptake in boiled and live neonatal humeri was analyzed over a time course of two hours. Arginine uptake increased linearly throughout the experiment and live humeri.
The boiled humeri did not exhibit a dynamic increase of arginine uptake in the experiment. The representative analysis shows the normalized and corrected arginine uptake. It is important to boil the contralateral bone as a active control.
This step is essential as the bone matrix may absorb radioactive amino acids, resulting in inaccurate results. The protocol has allowed us to confirm amino acid uptake kinetics both in vitro and in vivo.
