Actually I have two problem, First: I am using a chemotherapy drug on LNCap cell line(prostate cancer), and I expect to get a dose dependent result, meaning a decrease in cell viability by increasing drug concentration. But I see my highest concentration has the same viability with the lowest concentration. Why is it like this?
Second: I have done the test for several times, and each time the answer is different.
I have optimized every thing which could be effective.
Thanks for your helps.
Dear Samane, could you specify what parameters you have optimized? Have you established the linear range of the assay relative to the different number of cells in the well and the incubation time with MTT? Have you tried any control compounds, like staurosporine, which should definitely affect cell viability? How long do you incubate your cells with your compound and what is the range of concentrations of the compound that you are using?
Dear Samane
What concentration do you use?
How long is your incubation time?
How many times do you repeat each well?
What are OD used for read?
Dear Samane, usually I perform my MTT to 50,000/well, after 24 hoours, starts treatment and it take a certain time (your choice), after I put 10ul of MTT for 3 hours and finally dissoluting MTT's salts by DMSO or isopropanol-HCL (1 hour on incabator or 15- in shaker). Finally I mensure OD in 590 nm.
Remembering...somethings can be changes depending on what procotocol you're using!
Regards!
Darja Lavogina
Hello dear
About the cell numbers, yes I have tried seeding 5000 cells per well to 15000 cells, and seems that 8000 cell is ok, after seeding I incubate cells for about 44 hours (regarding to the doubling time of Lncap cell-line), and then I treat them with my drug by a range of concentration between 1nM to 2500nM(2.5 Micro molar) and I read them with MTT after 24h and 48h.
I have not tried any compound as control, yes it can be a test. Actually now I have decided to test my compound on another cell line to see if the problem is through my cell line.
Thanks for your help
Well, that seems reasonable, except that 44 h on plate seems quite long - usually, 24 h is enough. On the other hand, it also depends on how well adherent your cells are (cells that detach easily might be problematic). Do you change growth medium after 44 h? In addition, I would go up with the drug concentration to 10 microM or even 50 microM, if your stock enables that. Also, if your compound should rather affect proliferation of cells than cause cell death, you might get variable results dependent on the passage of cells. In fact, I strongly prefer resazurine assay to MTT, because you do not need to resolubilize resazurine and in our lab, resazurine assay has shown much better repeatability. Good luck!
your protocol is trued
i think your aliquot have problem it is not sufficient for your work
do you used the multichannel in your divide?
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