3H Thymidine Incorporation Assay for Rat-1a Cells: Difference between revisions
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'''Relevant papers and books''' | '''Relevant papers and books''' | ||
No further references are available at this time | |||
==Contact== | ==Contact== |
Latest revision as of 14:54, 24 June 2009
Overview
List of reagents is not yet complete
Procedure
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.
Notes
No further notes are available at this time.
References
Relevant papers and books
No further references are available at this time
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.