concentration of trypan blue... - please help me. (Dec/20/2006 )
hi all,
i am counting the no of viable cell. i am using the trypan blue. i use .4 % conc. of trypan but i am unable to differentiate between the living and dead cells.
kindly do the needful.
This is because your are using 4% trypan blue. Use 0.4%. Are you making your trypan blue solution in PBS? If not make it in PBS.
Hope this helps.
Thanx for your kind reply. i will do the as you told me.
I want a sugestion from i am studying inhibitory effect of one drug on my cell line.
This drug is having the inhibitory activity. and i wanna know i treat the cells, and after treatment some of the cell come out from and remain suspended in the medium.
I have plan like this.
1. Cell seeding in 24 well plate.
2. 24 hr incubation.
3. Then treated with the drug at various dilutions.
4. 24 hr incubation.
Now i will perform the trypan blue test some what like this....
1. Remove the medium from the well and then pellet down the cells.
2. Trypsinise the adhrent cells and then collect the cells. pellet down the cells.
I will do the trypan blue with both the cells.
Now i wanna discuss that am i going in the right way?
Is there any one who can suggest me something.
waiting for reply.
It depends on the cells that u r working on. Are those suspending or not? By the way the dose of trypan blue must b .2%. It means that u must add equal amount of ur stock trypan blue 0.4% to the equal amount of cell media.
Do you detach your cells which are not sloughed off from the monolayer? This is important because most of the cells in suspension will be dead. To determine % of dead cells you will need to have total cell count (dead+live cells).
i will have to work on the adherent cells.
dear exploresci, i wann ask from u that should i take the those cells which are suspending in the medium after treatment.
do u want to say that all the cell in the medium will be dead. so there is no need to do trypan blue test of test cell floating in the medium.
waiting for the response......
I suggest that you pool the medium and trypsinised cells in one tube, and then count, saves counting them separately and will give the proper live/dead ratio. It would be hard to calulate the proportions in the total population if you counted the sloughed cells and trypsinised cells separately.
I would also suggeest looking at some gene expression by western or real time PCR and depending on what the effect of the drug is, look at apoptosis and/or necrosis as well.
Then your work is so easy. U can add the equal emount of Trypan blue 0.4% to the equal emount of ur culture media to make 0.2% TB. Remove all the cell culture media and replace the TB solution instead of that. After 5 minutes remove this media and count the cells. TB can enter to the necrotic cells and make them blue, and the live cells are not blue. But remember TB just can detect the necrotic cells and not apoptotic cells. The cells that remained suspended were died at seeding time, then it isnt necessary to consider them.
Hope this helps.
Hi pBs,
As bob1 suggested it should be a count of adherent and suspension cells together not separately. It is not necessary that all the floating cells will be dead but it is most likely that dead cells will float in the medium.