3H Thymidine Incorporation Assay for Rat-1a Cells

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.

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