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

Overview

This video demonstrates an in vitro technique for estimating amino acid uptake by bone marrow-derived stromal cells. Na⁺-dependent amino acid transporters mediate the uptake of radiolabeled amino acids inside the cells, and the cellular uptake is estimated by measuring the radioactivity.

Protocol

The use of radioactive materials (RAM) requires prior approval by a designated safety committee at each institution.

1. Amino acid uptake in cells

1. 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.
2. Culture the cells for 2-3 days until confluent.
3. 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.
4. 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.
5. Aspirate 1x PBS and wash the cells once with 1 mL KRH.
6. 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.
7. Incubate cells with 0.5 mL of KRH containing 4 µCi/mL L-[3,4-³H]-Glutamine working media for 5 min.
8. 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.
9. 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.
10. 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.
11. Read radioactivity in counts per minute (cpm) using a Scintillation counter. Discard scintillation vials in scintillation vial waste container.
12. 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.
13. 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.

Materials

Name	Company	Catalog Number	Comments
0.25% trypsin	Gibco	25200
12-well plate	Corning	3513
Beckman LS6500 scintillation counter
Calcium chloride	Sigma	C1016
choline chloride	Sigma	C7077
D-(+)-Glucose solution	Sigma	G8769
DPBS	Gibco	14190
HEPES(1M)	Gibco	15630
L-[3,4-3H(N)]-Glutamine	PerkinElmer	NET551250UC
Liquid scintilation vials	Sigma	Z190535
lithium chloride solution, 8M	Sigma	L7026
Magnesium chloride	Sigma	M8266
MEMα	Gibco	12561
Microcentrifuge tube, 15mL	Biotix	89511-256
Potassium chloride	Sigma	P3911
Sodium bicarbonate	Sigma	S6014
sodium chloride	Sigma	S9888
Sodium dodecyl sulfate	Sigma	436143
Ultima Gold (Scintillation solution)	PerkinElmer	6013329
α-(Methylamino)isobutyric acid	Sigma	M2383