3H Thymidine Incorporation Assay for Rat-1a Cells

Overview

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Materials

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• supply 1 (i.e. tubes of a certain size? spreaders?)
• reagent 1
• X μL reagent 2
  • component A (reagent 2 is made up of multiple components)
  • component B
• equipment 1
• equipment 2

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

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References

Relevant papers and books

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1. Goldbeter-PNAS-1981 pmid=6947258
2. Jacob-JMB-1961 pmid=13718526
3. Ptashne-Genetic-Switch isbn=0879697164

</biblio>

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