While Iam doing MTT assay for seeding cells I am not using trypan blue. I would like to know if any problem will arise if I am not using trypan blue.
Thanks in advance.
Trypan blue is used to stain the dead cells. The cells that efflux out the dye i.e. they don't take up the blue stain would be your live cells and the cells that take up your trypan blue dye would be your dead cells.
You would require/use trypan blue to determine the number of live and dead cells per ml of medium.
Now the readings in MTT assay are based on the formation of formazaon crystals so it is neccessary that you have very precise seeding in your triplicates/quadruplates and throughout the other groups as well in order to see the change in the viability with your respective treatment.
The only problem that may arise would be in terms of not getting the absolute count (the cell number you thought you are seeding) and that may cause discrepancies in the results you thus obtain.
Hope this helps.
Hi Bhavapriya,
Yes it creates problem if you didn't count your cells using tryphan blue exclusion assay. The reason is whenever you are seeding the cells for MTT assay you have to make sure that your cells are healthy and viable (which will be determined by Tryphan blue assay). If there are too many dead cells then i wont setup my MTT assay till my cells become healthy.
Ram
Dear Bhavapriya,
It is a good laboratory practice (GLP) that you count your cell everytime with trypan blue, even in time of subculturing. Cell counting means, you count cells by using trypan blue, it is mendatory and keep record in your cell culture maintenance log book.
So that in any time, you have a record of cell status.
For MTT or any experiments, when you are going to seed cells, you need more than 95% cell viable (count via trypan blue, it is easy to do and well accepted), otherwise you can not proceed (it is recommended for cell line work in tissue culture).
Thank you
Good Luck!
MTT and other tetrazolium dyes depends on the cellular metabolic activity due to NAD(P)H flux. . It is important to keep in mind that assay conditions can alter metabolic activity and thus tetrazolium dye reduction without affecting cell viability.
MTT measure metabolic activity
Tripan Blue :It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue. A viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm.
Cynthia
When using a light microscope to observe adherent cells (PC 12) after an MTT assay I observe that the amount of blue precipitate is not distributed equally in the cells but that some cells are heavy blue whereas others contain almost no blue precipitate. Thus The value red after an MTT assay just gives you a mean value on a random population and you have to check the precise number of cells seeded using either a coulter counter ore to be more accurate the trypan blue assay which indicates that the cells seeded are truely live cells.
- Badges
- Science topic