I would like to add one query here about plate reading wave length. It is very confusing as some use 570 and ref 630, some other use 540 and ref 720, and also found 570 but no reference. So what is the ideal to apply for MTT assay plate reading? One literature mentioned that reference is not necessary but in his example, he uses reference too.
With regard to determine the number of cells to be seeded into each well of 96 well plate, I did growth curve too as suggested by others (refer the link below). However, I found that to get OD 0.75 to 1.2 after 3 days (reading day) from seeding (1st day seeding, 2nd day treating, 3rd day reading), it is impossible to have 80-90% confluency at day 2 on which to treat the cells. If we have to wait for cells becoming 80 to 90% confluency at day 2, thenOD of non treat cells will never be between 0.75 to 1.2 at day 3. If we chose to seed more cells number that give us 80 to 90% confluency at day 2 to treat the cells, the OD value is always over 1.2 and reached to 2.3 on day 3 (reading day) if no reference wavelength is set. The problem here is there are two catch points that contradicted to each other. This means, if we make 80-90% confluence at day 2 (treating day), we cannot get OD 0.75 to 1.2 at day 3 (reading day). If we want to seed less cell to have OD in that recommended range on day 3, cells confluency at day 2 never be 80-90% confluence. I notice if we can treat cells at about 40%, 50%, 60% confluency, it might be possible to have OD 0.75 - 1.2 at day 3 for non treated cells. So, anyone does treatment to cancer cells at 40 %-60% confluency only.
So, how do you guys adjust those.
Another thing that I noticed was that if we use 570 and ref 630, no matter how dark the color of the well, reader will give you max OD 1.5. Reader cannot distinguish darkness beyond the wavelength set unless you do not set Reference wave length. WIthout reference wave length you can get OD even fr 2.3 and over too.
So my question here is setting reference wavelength is good or not? Why? If not necessary, why do all others always set reference at 630 and why not 720 or others?
What cell density to use for MCF-7 and MDA-MB-231 MTT/MTS assay after treatment with common cancer chemo agents? - ResearchGate. Available from: https://www.researchgate.net/post/What_cell_density_to_use_for_MCF-7_and_MDA-MB-231_MTT_MTS_assay_after_treatment_with_common_cancer_chemo_agents#5797ab0396b7e4a0f47b0109 [accessed Jul 27, 2016].
Hi Jamail.
I have always used absorbance reading at 570 nm. Here is a link to a paper from Dr Jane Plumb whom I worked with, I believe she is the founder of the MTT assay.
http://download.springer.com/static/pdf/960/chp%253A10.1385%252F1-59259-406-9%253A165.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Fprotocol%2F10.1385%2F1-59259-406-9%3A165&token2=exp=1469704901~acl=%2Fstatic%2Fpdf%2F960%2Fchp%25253A10.1385%25252F1-59259-406-9%25253A165.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Fprotocol%252F10.1385%252F1-59259-406-9%253A165*~hmac=ed29a4f07d0e3d3bd721ef63398002cf768f6e012bc2755bbe8b69611818c33e
Hope this helps!
Most of the protocol use the Microtiter plate reader with and 570-nm and 650- filters .
If your machine is old or somehow unreliable, then use the ref wavelenght (either 630 or 720 or something between does not matter!). In some machines, the baseline may move upwards (e.g. from 0 to 1 OD) due to some electricity-related problems etc. You normally do not notice this! You only suspect it when your ODs are much higher than expected. In this case, you can overestimate/detect the OD by 1 OD (by mistake of course!). This is an artificial elevation and nothing to do with the cells reaction/the test itself. To correct this, ref wavelenght is helpful and it should be used along with the other wavelenght. The resulting OD value will be the OD 570 - OD 630. And this OD value is the right one! (attention: this is required in case of old machines or unstabile electricity supply problems etc) not all the time. If the machine is OK and as is the electricity, then do not worry about the ref wavelenght.
5.000 or 7.500 MCF or MDA is good enough for 48 or 72 h cytotoxicity studies. If you use the MTT as a proliferation assay then seed the less number of cells.Do not use DMSO as a slovent as it is used to be. It is a headache!. Use SDS with HCL. You can find the composition of this solution somewhere. Or ask me again. [email protected]
All the best
Engin
Thanks Rabbab Oun, Ravichandran Panchanathan and Engin Ulukaya.
Then if we seed 5000 cells it will become just about 10,000 after 24 hrs and this is not 80 to 90 % confluency yet to treat with drug. So, are you telling me that it does not matter? (Prof Engin)
Most of the papers, I don't know why they seeded 10,000.
However, for Phenotypic Microarray Biolog system recommended to seed 20,000 per well per 100 ul too. So what is actual determining facts to decide number of cells to be seeded.
All in all, Can we conclude that one can seed cells between 5,000 to 25,000 per well per 100 ul and no matter which number we seed, this number to be consistent?
With regard to OD setting. Reading is found to be same for all 570 nm. If reference is set to 630 nm, machine cannot read darker one and it just give its maximum OD 1.5. So no matter how dark it reaches to its cut off and OD will appear just 1.5. However, it can read lighter color and able to distinguish.
We can conclude for wave length like this. One should keep doing same setting no matter 570 and ref 630 or 570 and no ref or 570 and ref 720. Just stick to one setting. Is this fine?
No. 24 h after seeding, the cell number will more or less the same, even maybe little less!, but not the doubled! Maybe the next day, it can be doubled! We talk about the MCF-7 and MDA. There may be other cells proliferating so fast?! But this would not be usual actually. If the cell's size is small, then 10.000 cells may be OK. In the case of leukemia cells, even 100.000 cells can be seeded. Yes. One setting is fine! All the best.
Engin
Hi Engin, I have also been culturing MDA and MCF cells and noted clearly that hey are obviously more and never see less 24 hrs after seeding. If I am not wrong, it is about to double number of what I seeded. I checked online and noted their doubling time is about 24 hrs to 28 hrs. This is also different from what you knew. What I am telling here is passage 3 and above MDA or MCF cells. MDA and MCF cells are about 15 to 20 um in sizes. i did measure them. Some published papers said seeding for MTT should be decided according to the mitochodria activity. I am a bit confused now whether we have check the size of the cells or cells' activity. Thanks.
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