Radiolabeled Amino Acid Uptake Assay to Quantify Cellular Uptake of Amino Acids
Published: April 30, 2023
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 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 cultu…
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Etiketler
Bu Makaleden Alıntı Yapın
Radiolabeled Amino Acid Uptake Assay to Quantify Cellular Uptake of Amino Acids. J. Vis. Exp. (Pending Publication), e21318, doi: (2023).