3H Thymidine Incorporation Assay for Rat-1a Cells
List of reagents is not yet complete
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.
No further notes are available at this time.
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