[3H]ThymidineIncorporation Assay for Rat1a 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% TCAPBS0.5N 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.