[3H]CholineLabelingandTNFTreatmentofHL60Cells

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 2 X 105 cells/ml.

4) Add (0.5 µl/ml media) [3H] choline chloride to get ~0.5 µCi/ml.

--> Specific activity of the [3H] choline chloride is usually around 80 Ci/mmol.

5) Incubate cells with label for a period of 48 or 72 hours.

--> If experiment requires more cells then incubate longer or else incubate only for 48 hrs. Be consistent in the labeling period since cell cycle changes can have an effect on results.

6) Wash out label by pelleting cells and very gently washing 1 time with room temperature PBS.

7) Following wash, resuspend the pellet in 1/2 to 1/3 the original volume using prewarmed serum free media and count cells.

8) Seed cells into T-25 flasks for treatment: 10 mls/flask.

--> Thus, have 4.5 ml duplicates from each flask.

9) Allow cells to "rest" in incubator for 2-4 hrs (In a pinch, can leave overnight). Always keep track of rest time!!!

--> Equilibrate for longer periods to prevent controls from fluctuating.

10) Treat cells with 30 nM TNF in BSA for appropriate period.