Unfortunately, the highest wavelength in my lab's spectrophotometer is 492nm with a reference wavelength of 620 nm. From what I read, most published paper used 570nm wavelength to measure their MTT assay. I was wondering if that would affect my reading?
My seeding cell density is 9000 cells per well and incubate for 72 hours, after 72 hours add 10uL MTT, and incubate for another 4 hours, after 4 hrs, discard all media with MTT, add 100uL DMSO for 1 hr and read the plate.
The absorbance came out in a range of 0.1~0.4 (cells+media), 0.05~0.07 (media only, background).
I am doing MTT to check the cell proliferation when growing in different media formulation.
Should I increase my cell seeding density to 10000 per well?
It seems that in your case you get twice lower absorbance at 492 nm than you should get at 570 nm (where is the maximum absorbance of formazan, see the attachment). Reference wavelength is totally OK. It is not bad if you do not have other choice, until you measure the absorbance at the same wavelength in all cases including control.
Also it seems that you get really low absorbance (0.1-0.4) though you seed quite high number of cells (9000). Did you try to check through the microscope what was the confluency of your cells? If you seed too much, they may dye and then you get also low absorbance. I would suggest to do an experiment with different numbers of cells (2000, 4000, 6000, 8000, 10 000 cells/per well) and check which one will give you the highest absorbance after 72 h of incubation. Just always check the cell confluency through the microscope before the measurement. Good luck!
It seems that in your case you get twice lower absorbance at 492 nm than you should get at 570 nm (where is the maximum absorbance of formazan, see the attachment). Reference wavelength is totally OK. It is not bad if you do not have other choice, until you measure the absorbance at the same wavelength in all cases including control.
Also it seems that you get really low absorbance (0.1-0.4) though you seed quite high number of cells (9000). Did you try to check through the microscope what was the confluency of your cells? If you seed too much, they may dye and then you get also low absorbance. I would suggest to do an experiment with different numbers of cells (2000, 4000, 6000, 8000, 10 000 cells/per well) and check which one will give you the highest absorbance after 72 h of incubation. Just always check the cell confluency through the microscope before the measurement. Good luck!
Unfortunately, till now I did not have a study in this field.
I use comments. Thanks
With the best wishes
Mr Rezaei Ahvanooei.
I think it will compromise the data. If you are concernedabout total number of cells you can also use CellTiter 96® AQueous One Solution Cell Proliferation Assay System where you can measure at 490 nm. Promega, Catalog number selected: G3582. This is not advertisement but trying to give you a solution.
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