Do not remove the medium before adding MTT.

Yes, I think that might be the problem. You don’t remove the medium before adding MTT. Just add MTT-Solution (5mg/ml in PBS) in an amount equal to 10% of the culture medium volume (20µl to 200µl already in your well). Return plates to the incubator for 2-4 hours (You said: water bath @37C.... if you use CO2 free incubation conditions (L15 medium or such) thats okay. But if you are growing them in RPMI or MEM or DMEM they need to be incubated with CO2 atmosphere @37C) . Check after 2h if you can see black/lilac crystal needles or small clumps in nearly all cells of your positive control. If not wait longer (up to 4h) and check again. The incubation period depends on cell densities and cell metabolic activity. Afterwards remove the solution and add 100-200µl MTT solubilisation solution (I always use DMSO, works well for me. You just add it and shake the plate for 5-10 min @RT.).

Hope that helps.

Hi Palesa Send you a message,  Agree with the above answers.  In message I wrote out a step-wise system to check the various steps.  However I think the major problem is the MTT incubation.  Drop me a message if you want to give me a call.  I also think some of the people who moved there from Stellenbosch might have my contact details (You are at Farmovs I assume?)

Prof Pool yes am at Farmovs. I dissolved my samples in 20 ul DMSO and diluted with 1000 ul RMPI  medium and in my no compound control is only the medium.....Thank you all for your assistance, i'l give feedback...Thank You.

Hi Palesa,

Is it possible that the compound you are testing does not affect viability of H82 cells? Keep in mind that some compounds induce growth arrest and senescence but the cells are still viable, so you would not see any difference in viability using MTT assay.

In your next MTT experiment, it is a good idea to include a positive control, i.e., a compound that you know for sure will affect H82 viability (for example: 5% DMSO).

Hope this helps. Best of luck!

I think incubation between 2-3 hrs is enough after addition of MTT solution.

Hi Palesa,

Did you incubate plates inoculated with cells previously to add the compounds? (to reach 75-85% confluence)

Hope it helps you.

Palesa

You must add 20 ul DMSO to the 1 ml RPMI for the no sample control. This will exclude potential effects due to DMSO in sample solution.

Increase the cell number untill to 10000 cell each well and don't remove the media.

good luck

There is no problem with the method.

Did you check the cells under the microscope after aspirating the media?

DMSO can be used instead of MTT solubilasation solution to dissolve the crystals.

Good luck

I may point several potential problems here:

1. number of cells: seed 5000/10000 per well to get more robust response.

2. after exposure to the compound, wash it away by PBS before replacing medium

3. let them 12-24h in medium to recover after the exposure (optional)

4. add at least 50-100ul MTT medium (not PBS!)

5. solubilize in just 50ul of solubilization solvent (I use  acid isopropanol)

6. check your instrument for correct wavelength for readout

7. read out!

Advice: exclude outer wells from analysis due to so called "outer wells effect".

Wish you the successful exp!

Hello, are you sure that you have viable cells after 48h? Do you see any color at the end your assay? Have you checked your plate under the microscope? If you can see viable cells in your control  but not in your treated cultures, it is possible that your compound is highly cytotoxic at the given concentration. Besides that, you mention that you add 100μl of the compound under study (you do this 24h after seeding right?). What is the percentage of your compound's solvent in your working solution? If it is e.g. 10-100% DMSO or Ethanol, it is highly likely that your cells died because of the solvent's toxicity (if this is the case you will not be able to see any viable cells in your vehicle-treated (control) culture as well). You should prepare a working solution in such a way that the percentage of your solvent in every well (in the cultures treated with the highest concentration of your compound) will be less or equal to 0.1%. You can do that by preparing a stock solution using your solvent (e.g. 100mM) and then prepare all further dilutions using your culture medium.

How do you add and remove your solutions from the wells? If you are not careful during the addition/removal of your solutions there is a good chance that you detach/kill cells. You should tilt your plate at such an angle that you will be able to add or remove solutions from each well by placing your pippette tip at the side of the wells. You should avoid touching the bottom of the wells with your tip. 

Assuming that you have viable cells in your vehicle-treated culture and none of the above might be the cause of your problem have you tried seeding more cells? You can optimize your seeding density by testing 1000, 2500, 5000, 10000, 20000 cells/well. Cells should not be few but not too many as well. Empirically, after 48h there should be >70-

Hello!

I think the problem can be the low cell density. I usually use 50000cell/ml (25000cells/well in 48-well plate). In some cell lines I had to increase cell density to 70000cel/ml.

I hope it could help you.

Good luck!

The number of cells seeded per well is too low. Try increasing the cell density

If I understand you correct - you have removed all media before adding 20 ul of MTT solution. If so (no fresh media, MTT only) then your cells probably dried during incubation in water bath. What kind of media you used? Cultivation in whater bath is not the best conditions for the cells, esspetially if media CO2 dependent).

MTT problem is very popular topic ;) so, may be you will find helpfull information in folowing links:

https://www.researchgate.net/post/W..._is_my_MTT_Assay_not_turning_Purple

https://www.researchgate.net/post/D...MTT_assay_on_CSCs_that_I_can_follow

https://www.researchgate.net/post/Mtt_assay_suspense_cell_help_me

https://www.researchgate.net/post/P...ity_over_100_How_can_I_handle_this3

https://www.researchgate.net/post/What_is_the_OD_range_in_an_MTT_assay

If you have a problem with small cell amount or low metabolic activity than some other than MTT methods are available as well:

https://www.researchgate.net/post/In_XTT_proliferation_assay_what_is_the_role_of_PMS

https://www.researchgate.net/post/Does_someone_have_a_protocol_for_MTT_assay_on_CSCs_that_I_can_follow

https://www.researchgate.net/post/What_is_the_OD_range_in_an_MTT_assay

https://www.researchgate.net/post/What_are_the_zebrafish_embryo_derivatives

Helo,

the number of cells that you use should be enough, not too little or too much. Other pont is the time to plant cells and time of treatment, ie whether a cell population of 25% or 50% depending on the speed of cell growth and when the experiment was stopped.

good luck

Following corrections may help you..

--Number of cells should be increased, say ~4000-5000.

--After addition of MTT, plate should be kept in incubator (to perform enzymatic reduction of MTT efficiently)  not in water bath. 

--Be careful while removal of medium after MTT reduction. 

1. The cell number per well is too low, it should be around 4,000-10,000 cells/well for incubating 48 h with test compound.

2. Media containing MTT should be freshly prepared before assay, by mixing 1 vol of stock 10X MTT (5 mg/ml in PBS) with 9 vol of culture media, final is 0.5 mg/ml MTT in media. I use 100 microL/well, if your plate has 60 wells, 7 ml of the media containing MTT should be prepared by mixing 0.7 ml stock 10X MTT + 6.3 ml culture media. 

3. At the end of treatment, after you remove the media from wells, immediately adding 100 microL/well of the media containing MTT (0.5 mg/ml) and further incubating the plate in a CO2 incubator (not water bath) for 2-4 h (depend on cell line) to allow formation of purple formazan product. No need to wash the wells with PBS before adding MTT, it will wash out the weak cells away and you will get the over-cytotoxic results of your test compound.

4. To lyse the cells and solubilize the purple formazan product, remove the media out and add 100 microL of DMSO then shaking the plate on shaker around 3 min. Measuring absorbance at 550 nm by using a microplate reader.

Note: some cell line e.g. HT-29 die easily it you left it dry during you are removing media from the wells, so add the media containing MTT into the wells  immediately. But most of commonly used cell lines e.g. A549, HepG2, MCF7 can tolerate until you finish removing media from all wells in the plate.

I use this protocol for several years, you can look at in my paper. "Curcumin suppresses vasculogenic mimicry capacity of hepatocellular carcinoma cells through STAT3 and PI3K/AKT inhibition" Anticancer Res. 2014 Apr;34(4):1857-64.

Hello Dear 

There is no prob. in the method

 adding Compound in the prescribed cell line and incubate for suitable time than go through  cell count after 24 Hrs before adding MTT solution. 

It may be helpful for u 

Best of Luck