ThymidineIncorporationAssayforRat1acells

Thymidine-Incorporation Assay for Rat-1a cells      

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.          

  Reference   Conklin Lab
(http://gladstone.ucsf.edu/gicd/conklin/conklin.html)