I am trying to perform a MTT assay, and I have found some unexplained problems.
The protocol that we are using is the following:
1st day: Cells are seeded in 96 well plates (5000 cells per well) and incubated 24 hours.
2nd day: Wells are washed with PBS, and different concentrations of the materials are added, together with medium alone as control.
3th day: After a 24 hour incubation, medium with materials is retired, MTT is added (0.5 mg/mL per well) and plates are incubated for 3 hours. After that, MTT is retired, and DMSO is added to solubilize the formazan crystals.
When performing the experiment, we found that in some wells, apparently at random, cells in the centre of the well began to degrade, appearing with a round shape, and even membrane blebbing in some cases, while cells in the periphery remain healthy. It happened in different steps of the protocol: Sometimes, after adding the materials, other times in a moment between the addition of MTT and the addition of DMSO. This is particularly apparent when we check the cells after adding MTT, since you can see that the formation of formazan crystals is totally inexistent in the centre of the well. It looks as a toxic compound dropped in the centre of the well.
We tried changing the plates, and the result was the same: In some of the wells, this problem appeared. The cell line is growing well in the flasks, and after the first 24 hour incubation in the 96 well plates they always look healthy. This problem only appears after manipulating the plate.
I have worked before with this cell line, performing similar protocols, and I have never had this problem. Anyone can help me or give me any advice?
Thank you in advance.
are you sure cells doesn't wash off with PBS?In MTT assay I also had same problem in 24 wells.some random wells lost cells in the center. Later I realized after treatment of drugs made cell to ditched from the wall easily. During washing step be careful. add PBS slowly to wells and when removing too.
Dear Thilini Wickramasinghe,
Thank you for your answer.
I am sure that cells are attached in the centre of the well. I have checked the wells in all the steps. When this problem appears, I can see that cells are still there, but they have different morphology and they are not able to produce formazan crystals.
This is very interesting, what is the cell line you use? And also what is the amouth of medium in well?
Only explanation I am able to think about is the evaporation of media, as the medium evaporates and due to capillary effect is least medium in the middle of the wells...
Hope somebody with more experience will be able to answer this question.
Carlos Rumbo what is the FBS% in media? MTT was interfere with FBS.Our lab use 0.5%FBS+Media to treat drugs before adding MTT. We use 5ug/mL MTT solution followed by 3 hours incubation. Then dissolve crystals in DMSO. It is working well with lung cancer cells. Make sure it not get infection (Virus,Yeast etc.) during treatments.
Thank you for your answers,
Matej Vicen, I am using the A549 cell line, and we add 200 uL of medium per well. Moreover, we don't use wells from the periphery of the plate to avoid the edge effect. I worked before with these cells, performing similar assays, and I haven´t seen it before. I began to have these problem after arrive to a new lab, so the incubator is different, but culture conditions are the same (37ºC, 5% CO2).
Thilini Wickramasinghe, to incubate cells with materials, we use a 1% FCS medium. After 24 hours incubation, the medium with the materials is retired, and the MTT diluted in DMEM without FCS at 0.5 mg/mL and incubated for 3 hours.
I think that both evaporation and FCS concentration are not the cause of this strange effect, since it only happens in random wells, and not always. Cells are not contaminated during treatment, since we check the affected wells, and we didn´t distinghish any bacterial growth or turbidity.
Thank you for your help!
I have attached a picture of the appearance of a well with this problem. It corresponds with cells that were incubated 24 hours with DMEM 1% FCS. Then the medium was retired, and MTT was added for 3 hours. Cells in the peryphery (lower half in the picure) formed the formazan crystals, while cells in the centre of the well were unable to do so (upper half in the picture).
Those which produce no crystals are those which died when You washed them with PBS, it takes rather seconds than minutes under flow of air in laminar cabinet for the cell to dry out :-/ The edge between life and death is very sharp. Happened so many times before I realised ;-)
Adam Figarski
Thank you very much for your response.
I have worked before with this cell line, doing similar washes, and I have never seen that. In the past, I have used a higher number of cells per well (between 10000 and 30000), and now I am working with 5000. Maybe it can affect.
low amount of cells attract less water to stay in the bottom of well by cappilary forces, it fits together for me
Adam Figarski
Thank you for your response. I think that this is the most probable reason for that effect.
Thank you for your help.
Carlos Rumbo probably your cells are becoming dry when you are changing medium. I faced similar problem before. Try to be very fast when changing medium. I usually takeout medium and add medium within 3 seconds in each well.
Afsana Khanam
Thank you for your response. I agree with you. Finally, I could solve this problem being fast and letting cells without medium for the shortest period of time.
I have a different question, though. Whenever I seed cells in a 96 well plate, cells tend to grow towards the center of the wells. I always make a homogeneous cells suspension and mix well every time I add them to the wells.
Any suggestions are appreciated.
I have a similar issue, the cells in the outer well die while the inner wells are perfectly fine. and I don't think it is because of drying, because, I make a mixture of media and cells before adding a particular amount to the wells. In addition after adding DMSO, the cells on which I had added DMSO (minimum amount of 0.3ul) will die but the rest will be fine even though the compound is dissolved in the same DMSO. I am very confused and the issue is still persistent Can any of you give me any suggestions on what I might be doing wrong?
thank you.
that is way too long for 96well plate, the medium just dries out in outer wells. You can not use the sides, fill them with water or DPBS instead to protect inner wells . Or may switch to sth fancy ex.
https://www.businesswire.com/news/home/20160708005550/en/New-Cell-Culture-Plate-Eliminates-Edge-Effect-during-Extended-Incubation
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