Arachidonic Acid Labeling

1) Grow cells to a density of 5-8 X 105 cells/ml in RPMI 1640 containing serum.

2) Pellet cells and wash 1 time with room temperature PBS.

3) Resuspend final pellet in an appropriate volume of prewarmed serum free media and supplement with ITS (4 ml/L should yield 5 mg/L insulin and 5 mg/L transferrin).

--> "Appropriate" = bring cells to a final concentration of 5 X 105 cells/ml.

4) Add [3H] arachidonic acid to get ~2 µCi/ml (2 X 106 µCi/ml???).

--> Make sure final AA concentration is in the nM range.

5) Incubate cells with label for a period of 60 minutes (2 hours???).

6) Remove label by washing cells 3 X with ice cold media.

7) Aliquot washed cells to get 5 X 105 cells/ml again.

8) Treat with appropriate compound (and vehicle) for appropriate length of time.

9) Transfer 4.5 ml into each of two duplicate glass pyrex tubes and maintain on ice.

10) Spin down cells in the table top centrifuge at 1,200 rpm, 40C, for 5 minutes.