Cell seeding density - (Sep/29/2008 )
QUOTE (ambition @ Oct 2 2008, 06:58 AM)
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.
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.
You got 10,000cells per ml and you got 10ml in the T75flask.
This means you have a total of 10,000cells/ml x 10ml = 100,000cells
Then, you divided the cells into 12.5ml (because you need 1ml per well and the 0.5ml is for pipetting error)
100,000cells / 12.5ml
= 800cells/ml in each well
How to seed the cell?
1. if the cells are adherent, trypsinize them and neutralize the trypsin with medium+FCS. Then put the cells + medium in a 50ml tube
or
if the cells are suspension, collect them in a 50ml tube
2. spin at 800-1000rpm
3. remove the supernatant
4. add 12.5ml of your required medium to the cell pellet
5. gently resuspend the cell pellet with pipette
6. add 1ml of the medium containing the cell into each well of the 12 well plate.
7. put the 12 well plate in the incubator
Note: You can never pipette 1ml exactly, there is always a pipette error of ~±2 - 5%...therefore you need an extra 0.5ml in this case.
Hope this may help.
-Minnie Mouse-
Please check this thread for the cell culture protocol by Current Protocol (the golden standard in research)
http://www.protocol-online.org/forums/inde...showtopic=38932
-Minnie Mouse-