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| Author Information | |
| Primary Author | |
| Bruce Conklin ( bconklin@gladstone.ucsf.edu ) | |
| Affiliation | |
| UCSF/San Francisco General Hospital , United States | |
| Co-Author(s) | |
| Protocol Information | |
| Protocol ID / PID | |
| PUB128 | |
| Category | |
| Cell Biology | |
| Title | |
| [3H]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. | |
| Troubleshooting | |
| Reference | |
| Conklin Lab (http://gladstone.ucsf.edu/gicd/conklin/conklin.html) | |
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