3H Thymidine Incorporation Assay for Rat-1a Cells: Difference between revisions

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==Procedure==
==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.
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.
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.
* Seed 500,000 cells per well in a 24-well plate in DME + 10% calf serum.
    * Incubate 12–24 hours.
* Incubate 12–24 hours.
    * Rinse 1X with serum-free media.
* Rinse 1X with serum-free media.
    * Add 1 ml serum-free media.
* Add 1 ml serum-free media.
    * Incubate 24 hours.  
* Incubate 24 hours.  


2. Stimulating proliferation and labeling with [3H]thymidine :
2. Stimulating proliferation and labeling with [3H]thymidine :


    * Add drug. Incubate 16 hours.
* 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.
* Add 1 micro curie [3H]thymidine (NEN #NET-027Z) (1 ul diluted with 24 ul of media) to each well.
    * Incubate 8 hours.  
* Incubate 8 hours.  


3. Extraction of [3H]thymidine labeled DNA :
3. Extraction of [3H]thymidine labeled DNA :


    * Aspirate media.
* Aspirate media.
    * Wash carefully with 1 ml ice cold PBS.
* Wash carefully with 1 ml ice cold PBS.
    * Aspirate PBS.
* Aspirate PBS.
    * Add 1 ml of ice cold 5% TCA. Leave at 4 deg C for 30 minutes.
* Add 1 ml of ice cold 5% TCA. Leave at 4 deg C for 30 minutes.
    * Aspirate and wash once with PBS.
* Aspirate and wash once with PBS.
    * At room temperature, add 0.5 ml 0.5N NaOH/0.5% SDS.
* At room temperature, add 0.5 ml 0.5N NaOH/0.5% SDS.
    * Pipette up and down and add to scintillation vials.  
* Pipette up and down and add to scintillation vials.


==Notes==
==Notes==

Revision as of 14:53, 24 June 2009

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.

Notes

No further notes are available at this time.

References

Relevant papers and books

If this protocol has papers or books associated with it, list those references here. Below is an example for formatting purposes. See the OpenWetWare:Biblio page for more information.

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

</biblio>

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