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MTT assay problems, while testing anticancer activity


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#1 Ponybn

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Posted 28 March 2009 - 11:45 PM

Hi... I am testing anti cancer activity of a plant extract on jurkat cell lines. I performed mtt assay and the od values were taken at 490nm instead of the 570 mentioned (because of lack of facilities ) THe OD values included negative values at high concentrations of my drug(20mg) .

I am just not able to know why this problem occurs, am a rookie who is just interested in cell culture and cancer biology.

The procedure i followed was,
1) plate the 96 wells, and incubate for 24hrs
2) Add my extract at different concentrations and incubate for 24hrs.
3) Centrifuge and remove the supernatant
4) add mtt and incubate for 4hrs and dissolve in isopropanol
5) Read at 490nm

if there is any errors please suggest, also my extract is very viscous and thick like a paste, i dissolved it in water and DMSO separately, filtered them through 0.45micron syringe filter and used it.

My tutor says its the problem with the extract.

PLEASE HELP

#2 DRT

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Posted 29 March 2009 - 07:01 PM

When you say negative OD; do you mean that the reading for cells+media+extract was less than the reading for media+extract; or that there appeared to be an increase in cell viability?

Some compounds will directly interfere with the MTT assay, eg taxol. Also, high concentrations of coloured or poorly soluble compounds can make the MTT assay difficult due to the high backgrounds generated. With jurkat cells you should be able to centrifuge them down and carefully aspirate out the extract, replacing it with clear media before continuing the MTT assay. Your other options could be to use a cell counter or switch to a luciferase (ATP) assay.

Good luck, let us know when and how you get it sorted.

#3 Ponybn

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Posted 30 March 2009 - 10:10 AM

When you say negative OD; do you mean that the reading for cells+media+extract was less than the reading for media+extract; or that there appeared to be an increase in cell viability?

Some compounds will directly interfere with the MTT assay, eg taxol. Also, high concentrations of coloured or poorly soluble compounds can make the MTT assay difficult due to the high backgrounds generated. With jurkat cells you should be able to centrifuge them down and carefully aspirate out the extract, replacing it with clear media before continuing the MTT assay. Your other options could be to use a cell counter or switch to a luciferase (ATP) assay.

Good luck, let us know when and how you get it sorted.


The negative OD, is for cells+media+extract, the extract being in high conc of 15 and 20mg per well.

I suspect now that my extract is interfering and hence the variations in absorbance, so am planning to use just my extract in different concentrations in a few wells and check the absorbance.
so using this i can remove the error caused by this interference... ( i hope) i wil be doing this in a few days and will share it here...

Thank you, thank you, thank you......................

#4 DRT

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Posted 30 March 2009 - 02:58 PM

The negative OD, is for cells+media+extract, the extract being in high conc of 15 and 20mg per well.


That concentration seems very very high; 20mg per 200uL well = 200mg/mL?

#5 Ponybn

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Posted 31 March 2009 - 09:39 AM

The negative OD, is for cells+media+extract, the extract being in high conc of 15 and 20mg per well.


That concentration seems very very high; 20mg per 200uL well = 200mg/mL?


We made a stock by dissolving 10gms of our extract in 50ml of solvent. This was added as 10,15,25,50,75 and 100microlitres, which corresponds to 2,3,5,10,15 and 20mg. we could not get the activity of our drug in lower concentrations.

#6 DRT

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Posted 31 March 2009 - 06:41 PM

oops; :D sorry about the typo; 20mg/200uL=100mg/mL :angry:

We tend to consider anything that is not showing signs of cytotoxicity at 500ug/mL as being inactive; but this may not be relevant in your case.
Did all the extract dissolve properly or the 0.45um filter retain much of the extract? If not, it will be worth your while obtaining the dry weight of the soluble component that the cells were exposed to.

#7 Ponybn

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Posted 01 April 2009 - 09:07 AM

oops; :) sorry about the typo; 20mg/200uL=100mg/mL :lol:

We tend to consider anything that is not showing signs of cytotoxicity at 500ug/mL as being inactive; but this may not be relevant in your case.
Did all the extract dissolve properly or the 0.45um filter retain much of the extract? If not, it will be worth your while obtaining the dry weight of the soluble component that the cells were exposed to.



The extract dissolved nicely in water and ethanol. This was because it was extracted using the same solvents by the soxhlet apparatus. What did you mean by taking the dry weight of soluble component? how can i possibly do it??

#8 DRT

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Posted 01 April 2009 - 02:48 PM

The extract dissolved nicely in water and ethanol. This was because it was extracted using the same solvents by the soxhlet apparatus. What did you mean by taking the dry weight of soluble component? how can i possibly do it??


Drying, preferably freeze drying but anyway of evaporating off the liquid will work.

#9 Ponybn

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Posted 02 April 2009 - 08:54 AM

The extract dissolved nicely in water and ethanol. This was because it was extracted using the same solvents by the soxhlet apparatus. What did you mean by taking the dry weight of soluble component? how can i possibly do it??


Drying, preferably freeze drying but anyway of evaporating off the liquid will work.


I am attaching the OD values which i have got, I also have the layout with it. Please see it and guide me, i dont have any other source now :D

I took the OD values of only my extracts in the E and F row, cause i was getting negative values and i thought it was because of the extracts ( i think those two rows are a waste... , i just dont know ), also my extract is a crude one, it has not been purified by HPLC. I plated 150000 cells per well.

Please help,

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#10 DRT

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Posted 02 April 2009 - 04:19 PM

Has this plate been through the procedure you initially described above or did you skip the incubation steps? The cell controls with MTT look low, possibly indicating a problem with the wash at step 3.
Would it be an option to forget about your samples for a couple of weeks and look to validate your protocol with a dose response curve for something like Triton X 100? You may find for instance that you need to increase, or acidify, the isopropanol; and possibly the cell seeding rate is too high if jurkat's grow well. Then you can include a ‘standard curve’ on each plate when you start assaying your extracts (this helps prove to supervisors everything is working).
Unfortunately the only way I can think of for getting rid of that sample background, apart from switching to another assay, is to include a 2nd or 3rd wash step to completely remove the extract.

Keep at it, it will work eventually.

#11 Penguin

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Posted 03 April 2009 - 07:30 AM

Hi... I am testing anti cancer activity of a plant extract on jurkat cell lines. I performed mtt assay and the od values were taken at 490nm instead of the 570 mentioned (because of lack of facilities ) THe OD values included negative values at high concentrations of my drug(20mg) .


Are you sure it's ok to measure at 490nm?? This is 80nm away from the absorbance maximum of the purple formazan produced.

P

#12 Ponybn

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Posted 03 April 2009 - 07:54 AM

Hi... I am testing anti cancer activity of a plant extract on jurkat cell lines. I performed mtt assay and the od values were taken at 490nm instead of the 570 mentioned (because of lack of facilities ) THe OD values included negative values at high concentrations of my drug(20mg) .


Are you sure it's ok to measure at 490nm?? This is 80nm away from the absorbance maximum of the purple formazan produced.

P


hey penguin i am not sure whether we can do it at 490nm.. but i think it can it done.

#13 Ponybn

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Posted 03 April 2009 - 08:03 AM

Has this plate been through the procedure you initially described above or did you skip the incubation steps? The cell controls with MTT look low, possibly indicating a problem with the wash at step 3.
Would it be an option to forget about your samples for a couple of weeks and look to validate your protocol with a dose response curve for something like Triton X 100? You may find for instance that you need to increase, or acidify, the isopropanol; and possibly the cell seeding rate is too high if jurkat's grow well. Then you can include a ‘standard curve’ on each plate when you start assaying your extracts (this helps prove to supervisors everything is working).
Unfortunately the only way I can think of for getting rid of that sample background, apart from switching to another assay, is to include a 2nd or 3rd wash step to completely remove the extract.

Keep at it, it will work eventually.


The wash was done, i.e the supernatant was removed leaving the cell pellet and most of the sediments present in the extract also.. .well about washing with what should i wash? PBS? My tutor tells me that we cant wash the jurkat cells cause they are not adherent, i dont know if this is true.......

also are the formazan crystals formed from MTT soluble in PBS? what do u think from the results? does the extract have anti cancer activity? also i was told that the negative values show that cell proliferation is inhibited, is this true??

Thank you very much

#14 DRT

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Posted 05 April 2009 - 04:11 PM

The wash was done, i.e the supernatant was removed leaving the cell pellet and most of the sediments present in the extract


Sediments? I though your extract dissolved easily. I think you will always struggle with the MTT assay while you have extract sediment present. (unless it is possible to remove the solubilised formazan to a fresh reading plate at the last step while leaving all the cell/extract debris behind???).


My tutor tells me that we cant wash the jurkat cells cause they are not adherent, i dont know if this is true.......


It is true they are not adherent, which is why you centrifuge before aspirating off the media. This is a step where care has to be taken or your cells will end up in the waste.



also are the formazan crystals formed from MTT soluble in PBS?


No, that is what the isopropanol is for.

#15 Ponybn

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Posted 06 April 2009 - 10:40 AM

Thank you very much... i wil keep u posted of any upcomings... right now we are carrying out neutral red assay.. lets see how it comes up.. Thank you....... :rolleyes: :)




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