I am doing MTT assay for the drug in IMR-32 cell line. Initially I incubated the cells in 96 well plates for 24 hrs followed by drug treatment for 24 hrs after washing the wells with sterile PBS. After 24 hr treatment the media was removed and then MTT was added and incubated for 4 hrs. After dissolving the formazon the results we are getting is not in concordance with published data. Even after adding 5mM of drug there is no cell death at all where as published data showed 50 percent cell death.
Count and seed 1 x 105 cells in all wells and leave it overnight for cells to attach. Next day replace the medium with fresh media and then proceed with drug treatment. Incubate cells for 24 hrs, 48 hrs and 72 hrs after treatment and then check with MTT assay. Always keep six replicas for each concentration. Do not use PBS, use incomplete media for washing the wells.
Thanks for your suggestion. I have another doubt too. I usually prepare a master mix of drug and media for a total of 6 wells and then i distribute among the wells. Is it advised. Similarly I plate the cells. I count the cells and prepare a master mix for 70 wells and then divide them. Should I change the procedure and add in individual wells?
@Martin Kello: Thanks for your constructive comments regarding MTT. Adjusting initial cell number for MTT also important. I would like to know a little about grooving curve for cell number adjustment as you mentioned in your comments.
Hi Martin, Thank you very much for your details. I would like to see the phytochemicals effect on cell proliferation in ovarian cancer cells. I will see the effect after 24 and 48 hours of treatment. So to determine initial cell density suppose I am seeding 500-30,000 cells per well on day 0. On day1(24h) few wells will be~80% confluent. On day 2(48) some other wells with low number of initial cells will be ~80%. In your opinion which initial number will be better for the assay. If my question is not clear please let me know. Thanks once again for your prompt reply.
Is it possible to study the enhancement in cell viability by MTT assay due to the addition of some drugs?
Using multichannel Pipettes would improve the homogeneity of the elements you add (cells. drug, MTT...) and reduce the time difference between the wells in term of element addition.
Using freshly prepared FILTERED MTT (0.5mg/ml concentration) can improve results. Secondly, after 4 hrs incubation with MTT, remove MTT by multichannel pipette, and then add DMSO to dissolve formazon (100microlitre ) in each well and take readings instantly.
Do not centrifuge the plate in any step if your cells have adherence property.
I found a very good article: Assay Guidance Manual [Internet]: HTS assay validation, by Philip W. Iversen, et al
Link: http://www.ncbi.nlm.nih.gov/books/NBK83783/
I am still struggling to reach the error to comply with it.
If you could follow this paper, then your result would be consistent, even from lab to lab. If not... just forget to get the same result.
- Badges
- Science method