Thymidine-Incorporation Assay for Rat-1a cells

互联网2013-09-05

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Overview This method of Peter Coward, Ph.D. in the Conklin Lab was used in Coward, et al (1998) Controlling signaling with a specifically designed Gi-coupled receptor Proc. Natl. Acad. Sci. 95:352-357.
( http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9419379&form=6&db=m&Dopt=b )

More information from the Conklin Lab is available at ( http://gladstone.ucsf.edu/conklin.html ) Material DME + 10% calf serum

[3H]thymidine (NEN #NET-027Z)

5% TCA

PBS

0.5N NaOH/0.5% SDS Procedure 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.