You need 200,000 cells per well based on what you wrote, so in order to fill 30 wells (since you said you need 30ml total, with 1 ml per well), then that means you need a total of 6 million cells. You will divide 6.0x10^6 by your total cells/ml counted of 7.5x10^5. This will give you how many mls of cell suspension to take.

(6.0x10^6 total cells needed)/(7.5x10^5cells/ml) = 8 ml

30 ml total - 8 ml cell suspension = 22 ml media

I always make a little extra to make sure that all the wells are equally plated.

You need 200,000 cells per well based on what you wrote, so in order to fill 30 wells (since you said you need 30ml total, with 1 ml per well), then that means you need a total of 6 million cells. You will divide 6.0x10^6 by your total cells/ml counted of 7.5x10^5. This will give you how many mls of cell suspension to take.

(6.0x10^6 total cells needed)/(7.5x10^5cells/ml) = 8 ml

30 ml total - 8 ml cell suspension = 22 ml media

I always make a little extra to make sure that all the wells are equally plated.

From what I understand you want to seed 6 wells with 2x105 cells each (total of 1.2x106 cells) and you want to add 5ml to each of the wells (total of 30ml).

That means you'd need a solution with 1.6ml (out of your 10ml of trypsined solution) and you'd need to add 28.4ml (30-1.6ml) of medium to make up to 30ml.

I agree with Anne that it's always a good idea to make a bit extra: to have 25% extra have 2ml of your trypsinised solution and add 35.5ml of fresh medium.

You may also want to consider that cells will obviously grow so you can always use lower seeding cell concentrations if you're incubating your plates longer before treatment/collection.

that depends on the doubling speed of the cells,

I seed between 3000 and 8000 cells per well in a MTT assay,

Dear Mahitab, 

What I usually do for transfection and western blotting, I trypsinize and calculate the cells/mL. If I have x cells in 1mL and I want y cells for my experiment, then I would take y/x mL and spin it down. Now if I want to seed (y/x)/6 cells in each well in a 6 well plate, then I would resuspend y/x cells in 6mL media and distribute ImL to each well. I feel it'll reduce pipetting error. 

For sensitiviy assays I seed 1000 cells/ well. So for that I serially dilute  10^6 cells (to start with) 1:10 times to get 10^5 and again 10^4 then from that I calculate the total no. of cells I want. E.g. If i want to seed 1000 cells each in 12 well. I take 12,000 cells may be in x microliters, add (1200-x) microlitre media to it. Then distribute 100 microlitre in each well. 

Hope that helps!

thanks a lot for your advice and help

Dear Mahitab

I usually seed my cells in the 6 well plate in the concentration of 2*10^6 cells/well.

for the western blotting in which I can get more protien. So I use this equation

what you need/what you have * the final volume that you need

in your case it will be

/2*10^6/the number that you get after counting * 13 ml from this you will get how much cell suspension to take from your ( 10ml suspention) for example 3ml and 40 microleter 

then 13ml- 3.40 ml= 9.6  ml media

this mean you take 3.4 ml cell suspension+ 9.6 media= 13 ml total volume

from this you add 2ml to each well of 6-well plate 

I hope this would be ok .

good luck

Follow the instructions given by Anne von Koschembahr. As she says, prepare for at least 31 wells, otherwise you won't have enough for the last well.