Cell seeding density - (Sep/29/2008 )

Hello people,
I am new to cell culture and working with smooth muscle cells.
Can anyone tell me how to calculate seeding density of cells? To be more specific, say I harvest cells from T 75 flask and count is 500,000/ml. Now, how should I calculate number of cells per well if seeded in 12 well plates.
Please, help me out.
Thanks

Please check this post

http://www.protocol-online.org/forums/inde...ost&p=64847

Hey Thanks Minnie Mouse. The pdf is really good and helpful. But I want to know how to calculate number of cells per well. Anyone plz....

If you have 20ml in T75 flask, then you will have a total of 1x10^7 (500,000 x 20= 10,000,000)

If you seeded all the cells into 12 wells, then

1x10^7 cells/ 12wells = 833,333cells / well

I think it is too much cells per well.

Would you tell me the volume of the cell suspension in T75flask and the required seeding density in the 12 well plate?

The vol. of media in T75 was 10 ml. The cells were trypsinized, pelleted and suspended in 4ml of media. Counted by coulter counter and count was 500,000/ml......I want to put these cells in equal amount in each well in 12 well plates(1 ml/well) or say 96 well plates (200 ul/well)..What should I do?

Now, Minnie mouse you have divided the number of cells by 12well.....So is it no. of wells that matters or amount of suspension (in ml/ul)...

Thanks for your response, but I need more

....[The vol. of media in T75 was 10 ml. The cells were trypsinized, pelleted and suspended in 4ml of media. Counted by coulter counter and count was 500,000/ml......I want to put these cells in equal amount in each well in 12 well plates(1 ml/well) or say 96 well plates (200 ul/well)..What should I do?

hi,
i'll suggest what minnie mouse suggested but in other words:
you have 500.000X4ml=2000000 cells.
for 12well plates:
bring the cells from 4ml to 12.5ml.
then put 1ml in each well. discard remaining 0.5 ml. you'll have 166.666 cells/well

for96well plates:
bring the cells from 4ml to 20 ml.
put 200ul in each well. discard remaining 800ul . you'll have 20.8cells/well

hope I helped out

Hey Thanks man for your effort, but how you get 166.666 cells/well, and 20.8 cells/well. Can yo explain it little bit more....Plz...

[quote name='ambition' date='Sep 30 2008, 01:00 PM' post='152129']
Hey Thanks man for your effort, but how you get 166.666 cells/well, and 20.8 cells/well. Can yo explain it little bit more....Plz...

you have to pick a cell and then spit it in 1000 parts. then you pick 666 parts and put them in the well.
come on.....
it's just the result of the calculation .....it's like saying: in springfield 1.3men/10 are gay or 13men/100 or 130/1000.....and so on.

Please check this post for Sigma's cell culture protocol


http://www.protocol-online.org/forums/inde...ost&p=81417

Thank you guys, for your patience, may be its dumbest thing to ask but still its not very clear.
Let me put in my words, for calculating no. of cells per well, I have to divide number of cells per ml (say 10,000cells per ml) by the total amount of media (say 12.5ml for 12 well plate) required.....Is this the right way.