The use of radioactive materials (RAM) requires prior approval by a designated safety committee at each institution.
1. Amino acid uptake in cells
- Plate 5 x 104 ST2 cells in each well of a 12-well tissue culture plate. Plate cells in α-MEM containing 10% FBS, 100 U/mL penicillin and 0.1 µg/mL streptomycin (Pen/Strep). Plate extra wells of cells to quantify the cell number per condition for the normalizations in step 1.12. Incubate cells in a humidified cell culture incubator at 37 °C with 5% CO2.
- Culture the cells for 2-3 days until confluent.
- On the day of the experiment, prepare the following solutions: 1x Phosphate Buffered Saline (PBS), pH 7.4 and Krebs Ringers HEPES (KRH) buffer, pH 8.0: (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, NaHCO3, 5 mM HEPES, 1 mM D-Glucose). Prewarm to 37 °C.
- Aspirate the medium and wash the cells twice with 1 mL of 1x PBS, pH 7.4.
NOTE: This protocol is also appropriate for rapidly dividing non-confluent cells. In this case, it is important to normalize the radioactivity to either absolute cell number or DNA content. In addition, consider increasing the size of the culture plate or flask to increase the overall cell number and cpm values. This is important to determine empirically on an individual basis.
- Aspirate 1x PBS and wash the cells once with 1 mL KRH.
- Make 4 µCi/mL L-[3,4-3H]-Glutamine working media by diluting 4 µL of [1 µCi µL-1] L-[3,4-3H]-Glutamine stock per 1 mL of KRH.
NOTE: Before using radioactive materials, please contact the Office of Radiation Safety at your home institution to obtain approvals. All the procedures related to radiation must be performed behind the plexiglass shield.
- Incubate cells with 0.5 mL of KRH containing 4 µCi/mL L-[3,4-3H]-Glutamine working media for 5 min.
- Collect the radioactive medium and dispense into the liquid waste container. Wash the cells three times briefly with ice-cold KRH to terminate the reaction. Collect and discard all the washes in the radioactive liquid waste container.
- Add 1 mL of 1% SDS to each well and triturate 10x to lyse and homogenize the cells. Transfer the cell lysates to 1.5 mL tubes. Discard cell culture plates, serological pipettes, and pipette tips in the solid radioactive waste container.
- Centrifuge at >10,000 x g for 10 min. Transfer the supernatants to scintillation vials containing 8 mL of the scintillation solution. Mix by shaking the scintillation vials vigorously. Discard tubes and pipette tips in solid radioactive waste container.
- Read radioactivity in counts per minute (cpm) using a Scintillation counter. Discard scintillation vials in scintillation vial waste container.
- Trypsinize, resuspend, and count the cells from the remaining non-radioactive plates of cells (see step 1.1) 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. Normalize the cpm from step 1.11 to the estimated cell number from the non-radioactive plates.
- After completion of the experiment, decontaminate the cell culture hood, bench, and all instruments with a radioactivity decontaminant spray. Finally, perform wipe tests to ensure the working area is radiation-free.