I have used 25uM to 200uM concentrations of Thymoquinone (TQ) on MCF-7 (Breast Carcinoma), with 24 hours of incubation, but I didn't get proper cytotoxicity, I am going to increase incubation time as well as concentrations of TQ, but in articles, they have used 25uM - 150uM concentrations.
Concentration Selection is ok...But Incubation time should be 48 hrs...
U can cary different incubation times (24hr, 48hr and 72hr), with considering the cell's concentration in the well that u used. As some drugs are depending on time for it's activity.
you can try different thing, you can do time dependent viability assay. Compound that I am working on shows it's effect at 96 hour. And another factor that can change cytotoxicity is serum. you can treat MCF-7 cells in serum free DMEM or RPMI media after plating them overnight in 10% serum media. If you keep them in treatment for more than 48 hrs you need to change treatment media. In that case you are feeding cells with fresh treatment each 48 hrs. I hope this might help.
U can try the concentration at a range where it will give you reproducible result
doubling time of MCF-7 is 29hours so, increasing incubation time will be beneficial and as per dose selection is concerned, passage no. of the cells also influence the cytotoxicity. so variation in the results can be expected to some extent... and try to keep passage no. almost same (variation to 2-5 passage no. doesnot matter)...
kindly check these articles (may help)
http://link.springer.com/article/10.1007/s00280-010-1386-x/fulltext.html
http://www.sciencedirect.com/science/article/pii/S0006295211003637
Increase the incubation time period. First incubate for 24 hours if the result is not satisfactory incubate for 48 hours.
What should I do, If I have to add antibody with thymoquinone? Should I incubate antibody with TQ or I should go for antibody with 24 hours of incubation, then addition of TQ for the next 24 hours?
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