MTT problem, please help
Posted 08 October 2010 - 01:52 AM
I have been working with Caco-2 cells for a while and use them to do some MTT assay. I have some questions. Could anyone please help me?
1. How many cells are enough to do MTT assay? I tried 10,000 cells/well in a 96-well plate and incubated for 24hrs, but I didn't get much formazen.
2. Is there any better way to remove media and MTT solution without touching any formazen? I tried centrifuge before I took out the media, but I still couldn't remove the media without touching any formazen by using a pipette.
Posted 09 October 2010 - 06:52 PM
Posted 10 October 2010 - 01:25 AM
1. Incubate Caco-2 cells in a T75 flask until reach 80-100% confluence.
2. Dilute the cells to 100,000 cells/ml and add 100 ul/well to a 96-well plate, then incubate 24 hrs at 5% CO2, 37C
3. After 24 hrs, remove all the medium and add designed compounds, incubate for 4 hrs
4. Remove all the compounds and add MTT solution (5mg/ml), 20 ul/well, then 80 ul of Medium (DMEM with 10 % FBS, 1% NEA and !% penicillin)
5. Wait for another 4 hrs of incubation
6 After 4hrs, centrifuge the plate and remove the supernatant, then add 50 ul DMSO to dissolve the formazan
7. Measure the plate at 570 nm
I use DMEM, SDS and PBS as blank, negative and positive controls.
I have tried SDS/DMF solution to dissolve formazan and this can save the step of no.6, but I had problem to remove the colour from MTT solution mixed with DMEM, which affected my result significantly.
Posted 11 October 2010 - 11:07 AM
Posted 11 October 2010 - 03:18 PM
Posted 11 October 2010 - 05:08 PM
I am doing drugs screening. My compounds are peptide-based. I think 4hr treatment is sufficient to examine their toxicity. My senior strongly suggests me to incubate the cells at 50,000 cells/well for 3-5 days. He concerns that 24 hrs incubation of cell culture are not enough for cell adhesion to the plate surface. However, I wonder high cell number might affect sensitivity if the cells are over confluent . What I am trying to do is to modify this method to be more accurate and reduce error bar in the data, including limit lost of formazan, figuring out which cell number is sufficient for this method (it depends on different cell lines). Next time, I would like to try phenol red free medium to see if this can limit the interference of colour from medium.