I am experiencing very low O.D values in MTT assay, when I measured using microplate reader at 570 nm, even though I took enough no of cells there is no much increase in O.D. is it quite common or some error (please answer w.r.t. MCF-7 cells)
best solvent is DMSO
and OD range is roughly .05 to 0.7-0.8 ; outside this range it is lesslikely to be propotional to cell number
dear bomme
will u plz tell how much cells you are culturing per well?
at what time interval you are taking O.D? Normal range for MTT say is 450-500nm. reference absorbance at a wavelength of 630-690nm.
Hi
The value of OD will be depend on the amount of cells per well. Its means if you had small amount of cells per well you will receive the low OD.
According to Mosmann T. (Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.) you need use the OD 540 nm.
You measure not MTT, but formazan which dissolved into DMSO and has a purple color. The range of absorbance of formazan is between from 400 to 650 nm with peak at 540 nm. You could use different wavelength in this range it will depend on of your spectrophotometer
many factors required such as number of cells cultured/ per well, type of cell adherent or suspension
Normal range for MTT say is 450-500nm.
Hi all, Thanks for your answers, cell number I took was 2x105. Time of the interval is 4hr. Could anybody suggest me the best solvent for formazan dissolution?
best solvent is DMSO
and OD range is roughly .05 to 0.7-0.8 ; outside this range it is lesslikely to be propotional to cell number
best and most rapid solvent is DMSO
we are using DMSO:Etanol (1:1) for adherents cells
optimal OD level is determined by features of spectrophotometric measurements and best in range of 0.2-0.8, but in practice could be used OD levels from 0.05 to 2
If sensitivity of MTT assay not good for you you may try other metabolic tests.
MTT problem is very popular topic ;) using search option may be usefull.
So, may be you will find helpfull information in folowing links:
https://www.researchgate.net/post/Why_is_my_MTT_Assay_not_turning_Purple
https://www.researchgate.net/post/Does_someone_have_a_protocol_for_MTT_assay_on_CSCs_that_I_can_follow
https://www.researchgate.net/post/Mtt_assay_suspense_cell_help_me
https://www.researchgate.net/post/Problems_with_IC50_determination-cell_viability_over_100_How_can_I_handle_this3
Thank you, Danill R.
I will try this as you said my O.D range starts with 0.05, I thought it is too low. Anyhow I will repeat the experiment.
Do you mind sending me a reference referring to optimum OD for MTT is 450_600 ?
All the references I have say from 500-600 . I only have 450 nm and 620 nm in my lab .
Dear Mohamed,
If you have set of two filters 450 and 620 nm only then use DMSO (or DMSO:EtOH 1:1) to solubilise MTT formazan prior to measure signal (instead of acid isopropanol). It give you better signal/noise ratio due to differences in spectra.
For details take a look this publication http://link.springer.com/protocol/10.1385%2F0-89603-282-5%3A137
Daniil R. Petrenyov: regarding to your answer. may i know any article discussing about that? i need to know for citing in my article. TQ
Dear Fazirah Rosli sorry I don't have saved source of our protocol.
Mentioned above protocol could be cited (http://link.springer.com/protocol/10.1385%2F0-89603-282-5%3A137)
additional information about solvent for MTT assay could be found here https://ru.scribd.com/doc/241231597/MTT-assay-Protocol
As for me mix of DMSO and Ethanol more rapid, but isopropanol less expensive.
As I can understand OD spectra of formazan depends of pH thus using of mentioned solvent give us same results in same pH conditions.
one more tread to read: https://www.researchgate.net/post/DMSO_versus_isopropanyl_alcohol_for_MTT_assay2
MTT Cell Proliferation Assay from ATCC they recommend the good cell concentration for MTT fall in an absorbance of 0.75-1.25
http://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx
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