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

Abstract

Source: Shen, L., et al. Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts. J. Vis. Exp. (2022).

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, p....

Materials

List of materials used in this article

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