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Best method to calculate IC 50 MTTv/s clonogenic assays - (Oct/16/2007 )

Hi,

I am trying to find the IC 50 for some newly systhezized platinum drugs in cancer cell lines. I have been doing the colony growth assay where I am seeding the well plates with 500 cells and the use different concentrations of the drug . I am exposing the cells to the drug for 1 hr and then changing the medium and keeping the plates undisturbed for 7 days . After 7 days I am staining the plates with crystal violet and then counting the visible colonies having more than 50 cells. Then by performing linear regression calculating the IC -50. The values of IC 50 which i am getting with this method are higher. This was tested by doing the same assay on Cisplatin and carboplatin .

There are many labs which are using MTT assay for calculating the IC 50 . I would like to know the advantages and disadvantages of doingthe MTT assay .IF anyone is using this kind of clonogenic assay please let me know if you are facing the same problem .

which of the two assays MTT v/s clonogenic is more reliable.

Please help.

Thanks ,

SRJ

-Srj-

Hi SRJ,

in my opinion not being an expert in this field both tests have their pros and cons, the pro for the MTT test being that it is somewhat faster than your assay. But I have another question - I am pretty sure that it makes much more sense to use non-linear regression (sigmoidal doseresponse) to calculate IC50 values.

kr├╝mel

-kr├╝melmonster-

Hi
i want to add these points-
1. MTT is fast rapid and based on the metablic status of the cells (contition of cell is imp factor)
2. CA is slow, and is based on the colony formation capacity of the cells (so the cells is imp factor)
3. the compounds u r testing are getting analysed for two different time point ( i mean in MTT it is just 24-48h nd in CA it is more than /days)
U might get a variable figures as these assays have their self importance and which is conditional.

-adozelesin-

QUOTE (adozelesin @ Oct 25 2007, 01:49 AM)
Hi
i want to add these points-
1. MTT is fast rapid and based on the metablic status of the cells (contition of cell is imp factor)
2. CA is slow, and is based on the colony formation capacity of the cells (so the cells is imp factor)
3. the compounds u r testing are getting analysed for two different time point ( i mean in MTT it is just 24-48h nd in CA it is more than /days)
U might get a variable figures as these assays have their self importance and which is conditional.


I'm wondering if you'd be willing to expand these 3 points because I'm having trouble repeating results obtained through an MTS assay in clonogenic assay. In a MTS assay, I observed that my cells displayed 100% viability when exposed to the highest concentration of my drug of interest over 72 hours. However, when I exposed them to only 24 hours of that drug at that highest concentration followed by 21 days of growth (in non-drug containing growth media) in a clonogenic assay, there were significantly fewer colonies (relative to the untreated control).

Do you have any potential explanations for this difference in cell behavior? Could it be that (in the clonogenic assay) after 24 hours of exposure, the cells become cytostatic and simply no longer replicate and form more colonies, but that in the 72 hour MTS assay the cells are seen as 100% viable because the assay does not test their potential to form colonies but rather only reflects viability? I'd appreciate any insight you may have. Thank you in advance.

-shingouki-

I have a quick question relating to colony assay , i want to know how you seed your cells , whenever i i need to plate 500 or 600 cells i always have a problem as my original sample has more cells so how do you dilute yours to get the cell number you need. Can you explain in detail. Would be great. Thanks:)

-seashell83-