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proliferation assay - labeling cells with 3H thymidine (Aug/30/2004 )

hi! for one month i've tried to get significant results when performing a 3H thymidine incorporation assay on chicken granulosa cells to show that the protein i work on enhances proliferation of those cells. i always synchronize the cells in a low growth state, add the protein, incubate for 18 hrs followed by a 6 hrs thymidine pulse and then i use 5% TCA and 0.5N NaOH/SDS to solubilize the cells and measure the CPM/DPM in a scintillation counter. the problem is, i get a maximum of 200 counts (background? the negative control - untreated cells - is around 100 counts) and my boss thinks this could be because the TCA/NaOH method of getting the DNA out of the cells is too "dirty" and all the cell stuff negatively influences the result. does anyone have suggestions or know a proliferation protocol that is working?


Dear Roxy, I haven't a big experience in thymidine incorporation but in my institute people do in this way:
- 24 well-plate
- Add Thymidine to each well in order to obtain a concentration of 2µCi/ml , six hours before the end-time point chosen. (6+18)>>>24 hours.
- By working at room temperature, aspirate medium and rince wells with 1 ml of PBS without Ca and Magnesium.
- Add 1ml of 5% TCA. Leave at +4°C for 30 minutes.
- Wash with 70% Ethanol
- Leave to dry it
- Add 0.5 ml of 0.2 N NaOH and leave it at 37°C for 30 minutes
- Leave at 37°C for 30 minutes
- Collect and put all in 7 ml of Instagel. Wait four hours before put in the Betacounter

Good luck...Let me know if it works...


Other methods/kits should also suit for your purpose - cell proliferation assay in addition to H3-thymidine labeling, like the following quantitative assays:
(1) MTT, XMT, WST-1, WST-8, CCK-8.....
(2) BrdU labeling or PCNA labeling.....