3H Thymidine Incorporation Assay for Rat-1a Cells

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This method of Peter Coward, Ph.D., was used in Coward, et al (1998) Controlling signaling with a specifically designed Gi-coupled receptor Proc. Natl. Acad. Sci. 95:352–357.

1. "Making cells quiescent," i.e. synchronizing the cells in a low growth state.

  • Seed 500,000 cells per well in a 24-well plate in DME + 10% calf serum.
  • Incubate 12–24 hours.
  • Rinse 1X with serum-free media.
  • Add 1 ml serum-free media.
  • Incubate 24 hours.

2. Stimulating proliferation and labeling with [3H]thymidine :

  • Add drug. Incubate 16 hours.
  • Add 1 micro curie [3H]thymidine (NEN #NET-027Z) (1 ul diluted with 24 ul of media) to each well.
  • Incubate 8 hours.

3. Extraction of [3H]thymidine labeled DNA :

  • Aspirate media.
  • Wash carefully with 1 ml ice cold PBS.
  • Aspirate PBS.
  • Add 1 ml of ice cold 5% TCA. Leave at 4 deg C for 30 minutes.
  • Aspirate and wash once with PBS.
  • At room temperature, add 0.5 ml 0.5N NaOH/0.5% SDS.
  • Pipette up and down and add to scintillation vials.


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