Dear Himanshi,

If you add MTT directly and your drug has influence on cellular enzymes activity (enhance or decrease) you will have falsified results (positive or negative). Therefore in my opinion it is better to remove drug before MTT addition.

Dear Himanshi,

In MTT assay it is not necessary to change the media, you have to add MTT solution to the existing media, and incubate for further 3-4 hrs and then remove the media and dissolve with DMSO. Finally after shaking, your plates are ready for reading by plate reader and detecting the absorbance of the viable cells.

You should incubate the cells for 72h in culture media and then check the viability with MTT.

After 24hr,48hr and 72 hrs of treatment were finished, please remove the medium and then add freshly prepare MTT solution to incubate 4 hrs@37 C . next remove the medium to add 50 microlitre DMSO to shake 1 minutes to read at 570 nm. Best of luck

Thank you all for your suggestions..

Dear Himanshi,

I don't know about others but I'm telling you from my experience...

1. After drug treatment wash the cells with PBS twice.

2. Then resuspend the cells in 180 µl PBS + 20 µl of 5 mg/ml MTT and incubate it for 4 hrs at 37 degree C.

3. remove MTT and resuspend in 200 µl DMSO to dissolve insoluble formazan, giving a purple color.

4. measure the absorbance of the samples.

Thanks...

As Kamalika said, after incubating the cells treated with the drug, remove the serum containing medium. Dilute the MTT stock solution in serum free medium, add and incubate for 4 h.

Dear Himanshi,

If you add MTT directly and your drug has influence on cellular enzymes activity (enhance or decrease) you will have falsified results (positive or negative). Therefore in my opinion it is better to remove drug before MTT addition.

Himanshi, we usually incubate the cells in Hanks balanced salt solution / Krebs Ringer buffer while carrying out MTT assay ie., MTT working solution is prepared in any of these buffers

I think once you finish the drug treatment, remove the drug containing medium, wash cells twice with pre-warmed PBS, then add the MTT diluted either in HBSS or desired culture medium without serum, incubate them at 37°C for 4 hours, remove the MTT solution, wash cells with pre-warmed PBS twice, add DMSO, shake for a while and read the plate at 570 and 690 nm.

Hope this helps! Good Luck!

Hi, having personal experience, I will go with Sandip Pal and Abhijit Shinde. Remove the serum and drug containing media which may affect enzyme activity to reduce MTT. After the drug treatment is over, remove the media, wash the cells with PBS and then add MTT, incubate them in humidified incubator at 37°C for 4 hrs. Remove the MTT solution, wash the cells with PBS and then add DMSO to dissolve formazan. Measure the absorbance.

Alternatively, use MTS (one step MTT assay). To do so, remove the drug containing media and directly add MTS solution and measure the absorbance. All the best.

Option # 1. At the end of incubation, remove the media and replace with fresh medium as before. As suggested by others in this forum, add MTT and perform the assay. That way you are measuring the cytotoxicity at the end of the treatment period. During your MTT assessment, the assay is not being interfered with by the drugs/treatment in the wells.

Option #2. Alamarblue assay is similar to MTT and is simple to use. Add 10% v/v of alamarblue dye to the wells and read the OD570-600nm. In this you could test this by both ways. With washing off the drug and without washing. If the difference is negligible or none, keep it simple for this class of drugs. Thus, it is context specific. If the drug you are using is not mitochondria targeted you may not see much with your washing procedure.

Hope this helps.

What I usually do is to wash the cells with fresh media once and add MTT prepared in media to start the assay. It worked well.

Hi Himanshi,

There is some good advice already in the other answers. The procedure I use is:

Remove the media.

Wash cells with PBS.

Add a 0.5 mg/mL solution of MTT (100 µL per well for 96-well plates).

Incubate at 37C (the optimum time depends on the cell type - typically 1 hour for cancer cell lines or 2-3 hours for primary cells).

Remove the MTT solution.

Add 40 µL per well (for a 96-well plate) of 0.1 M HCl in isopropanol.

Cover with plate lid and wait until all formazan dissolved.

Measure absorbance at 570 nm with background at 630 nm.

You could also add the MTT made up in fresh media instead of PBS, as Ling suggests. It's rare but I have had experience that some cell types don't like being left in PBS for a while and can detach from the plate, in which case you have to add MTT in fresh media.

Best of luck!

Mark

Yes, change the medium if drugs interfere with the reaction, exemple sbV antimony strongly interfere with the reduction of the salt.

I would prefer MTS over MTT as it is simple, one step precedure.

Dear Asavari, could you please attach your MTS protocol here?I though CCK-8 should be a better option but it is not available in my lab now. 

Hi, 

i wouldn't wash cells on 96well plate since they don't detach (not saying it's wrong, but I prefer not to interfere) but definitely I would dissolve the reagent in your fresh culture media w/o phenol red and antibiotics Before the incubation. MTS assay is indeed easier but it's more of a  viability test than proliferation (MTT sometimes is treated as a proliferation test- I don't agree on that but it's very cheap and as a tool before checking apoptosis etc. Is good enough). So depending on what you want to prove You should pick the test. cCk-8 test has a really broad range of detection, so that would be definitely a MTT test I would go for. If you're searching for an MTS kit, I'd reccommend Promega's kit i used many times.

http://fi.promega.com/products/cell-health-and-metabolism/cell-viability-assays/celltiter-96-aqueous-non_radioactive-cell-proliferation-assay-_mts_/

Hi Milena Doroszko,

You seem quite experienced with MTS,I have some querry regarding it.

1. Is above mentioned procedure (fresh media w/o phenol red ) is applied for MTT or MTS too? Promega says add 20% MTS reagent to each well. Is is necessary to use 20% or we can lower. I use 10% MTS ( and result seems okay. but i am not sure, is that fine or not.

2. Regarding incubation after adding MTS, 1-4 hr, how can we confirm how long for which cell lines should we incubate for?

Thank you

Hi Kamal,

I would exchange the medium in both, MTT and MTS. You must realize that the evaporation in the external wells is quite significant and that your treatment might change MTT/MTS color. I usually leave the outer wells without cells but with media (have my cells B2-G11), do control treatment in 2 columns (different places) to ensure the reliable quantification. So after the incubation time I would blot the plate and fill it with DETECTION REAGENT (fresh medium (warmed up) without phenol red, without antibiotics, containing 20% MTS) . If you have little cells, you could decrease the volume of detection reagent, but not medium/MTS ratio- sometimes I did that myself when running out of reagent (decreasing the volume 50-100ul). Of course it's best if you check your cell lines first as some cells naturally produce more formazan, therefore decrease in the MTS volume will not be possible. It's colorimetric method and it has its own dynamice range and as long as your cells won't reach the edge of the range everything should be fine. In all of cells I checked (around 30 types of different human and mouse lines) 3.30h was quite ideal. You might check the A after 2h also and compare them- I've found that for cells that produce a lot of formazan after  4h the absorbance is higher in all wells, but result do not differ between 2h and 4h (as treated groups are normalized by control wells). With new cell line I do measurement after 2h and the end-point at 3.30-3.45h, afterwards only the latter. Cells must be all the time kept in 37C though.

If you have any other questions I will be happy to help :)

I tried both ways. You can eaither change the medium, or you can add the reagent to the existing old medium.

 I agree with professor Sławomir Lewicki because in our lab we are all trained like that only.  

Better you remove your media containing the drugs before adding MTT. Make sure that you make a vehicle control (the medium in which the drug is dissolved per se DMSO) to check the effect of Dissolvent of your drug in the cells. The amount of signal generated is dependent on several parameters including: the concentration of MTT, the length of the incubation period, the number of viable cells and their metabolic activity. All of these parameters should be considered when optimising the assay conditions to generate a sufficient amount of product.

I think it is better to remove drug before MTT addition.