Radiolabelled nutrient uptake for suspension cells

Radiolabelled nutrient uptake for suspension cells (eg T lymphocytes)

Amino acid uptake buffer (AA uptake buffer)

HBSS (Gibco cat#14025092) with 5mM non radiolabelled amino acid (eg leu, gln, phe)

Amino acid tracer buffer

            2mCi per sample in 200ml AA uptake buffer

Glucose uptake buffer

            Glucose free RPMI (Gibco cat#11879020)

Glucose tracer buffer (or 2-DG tracer buffer)

            1mCi per sample in 200ml Glucose free RPMI

Oil mix = 1:1  Poly(dimethylsiloxane-co-methyl-phenylsiloxane) : diisononyl phthalate

* Poly(dimethylsiloxane-co-methyl-phenylsiloxane) viscosity 125cST - Sigma-Aldrich cat# 378488

* Diisononyl phthalate – Sigma-Aldrich cat#376663

Count cells.  It is critical to have an accurate cell number/count before starting.

Uptakes are performed in triplicate per biological sample

You will need 2x10⁶ cells per uptake, so 6x10⁶ cells per biological sample.

         (It is possible to do with fewer cells ie 1x10⁶, but need to work out sensitivity over background for each cell type. Work this out by performing uptakes on a serial dilution of cells)

Set up the uptake assay prior to harvesting cells.

1. For each uptake sample, layer 0.2ml tracer buffer over 0.5ml oil mix in a 1.5 ml Eppendorf tube.

2. Spin the tubes at 6000-7000g to settle layers.

3. Resuspend cells at 10x10⁶ per ml in uptake buffer ie 2x10⁶ cells in 0.2 ml uptake buffer

4. Add 2x10⁶ cells in 0.2 ml uptake buffer to the tracer buffer layer on top of oil mix.

5. After a 3 min tracer-uptake period (room temperature @19-21C), spin down at 6000-7000g for 4 mins to pellet the cells below the oil, thereby terminating the uptake of radiolabeled solute.

         (This time point was worked out for in vitro cultured murine cytotoxic lymphocytes and uptake was linear from 1 min to 6 mins under these conditions – for other cell types, we would recommend performing a similar time-course experiment, eg 1 min to 20 min to determine optimal tracer-uptake period.)

6. Aspirate the tracer containing aqueous supernatant from the top of the oil layer (leaving the cell pellet undisturbed at the bottom of the tube).

7. Rinse any remaining radioactivity from the tube walls with water and aspirate from the top of the oil layer (leaving the cell pellet undisturbed at the bottom of the tube). Repeat.

8. Aspirate final water rinse and oil mixture, leaving the cell pellet undisturbed at the bottom of the tube.

9. Lyse and resuspend the cell pellet in 0.2ml NaOH (100mM).

10. Transfer the 0.2ml sample to scintillation vials.

11. Add scintillant suitable for aqueous solutions (we use 3mls of Optiphase HiSafe 3, Perkin Elmer)

12. Measure β-radioactivity in scintillation counter.

Controls to include

         No cells : to control for background radioactivity following through washes and transfer from tubes to scintillation vials)

         Quench : 10mM non radiolabeled amino acid / glucose to specifically compete with radiolabelled tracer

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