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cytotoxicity screening problem using MTT assay


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#1 msfive

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Posted 11 April 2010 - 07:41 PM

Hi! I am currently screening for the cytotoxicity of some bacterial crude extracts on MCF-7. However, I am having problem with my results. Most of the extracts obtained a 100% survival and also most have higher % survival in high dose than in low dose. the high dose is 50 ug/ml while the low dose is 5 ug/ml. all treatments for every extract is in triplicates. moreover, in my positive control (cells treated with doxorubicin), my low dose (0.0004 uM) obtained more than 100%. i computed for the standard deviation of each treatment and the result was low (range of 0.002-0.07 deviation).

my procedure:
1. seeding of cells in 96-well plate with 20,000 cells/well (200ul/well), incubate
2.after 14-16 hr, treatment of cells with crude extracts in triplicates (high dose= 50 ug/ml, low dose=5 ug/ml)
3. after 72 hr, remove media, add 15 ul MTT, incubate
4. after 3 hr, add 100 ul DMSO
5. read at 590nm

so, my questions are:
1. is this kind of results possible especially on my positive control?
2. if not, is the problem due to my seeding of cells?
3. is there something wrong in my procedure?

many thanks!

#2 Inmost sun

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Posted 20 April 2010 - 07:15 AM

Hi! I am currently screening for the cytotoxicity of some bacterial crude extracts on MCF-7. However, I am having problem with my results. Most of the extracts obtained a 100% survival and also most have higher % survival in high dose than in low dose. the high dose is 50 ug/ml while the low dose is 5 ug/ml. all treatments for every extract is in triplicates. moreover, in my positive control (cells treated with doxorubicin), my low dose (0.0004 uM) obtained more than 100%. i computed for the standard deviation of each treatment and the result was low (range of 0.002-0.07 deviation).

my procedure:
1. seeding of cells in 96-well plate with 20,000 cells/well (200ul/well), incubate
2.after 14-16 hr, treatment of cells with crude extracts in triplicates (high dose= 50 ug/ml, low dose=5 ug/ml)
3. after 72 hr, remove media, add 15 ul MTT, incubate
4. after 3 hr, add 100 ul DMSO
5. read at 590nm

so, my questions are:
1. is this kind of results possible especially on my positive control?
2. if not, is the problem due to my seeding of cells?
3. is there something wrong in my procedure?

many thanks!


the amount of doxorubicin seems to be very low, I doubt thatt a significant increase of apoptosis is to detect;

the best is to re-start the experiments to show if the bacterial extract really strengthens the survival of cells; it is not irrealistic to observe an increase of survival if signaltransduction pathways of survival are triggered by the bacterial extract...




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