Preparing cells for plating - (Jul/22/2013 )
anyone can help me about this..
Let say I want to plate cells in 96 well plate as follow:
-------------> 100 µl of cell suspension in one well . and each well should have 10000 cells
-------------> i need 7 ml of cell suspension (some wells not use)
-------------> how much cells i need overall?
-------------> anybody can show me how i going to do , when i count cells, the cells are too many , and what if they are less..can anayone giv eme example
-----------> how i going to sure that after i plating the cells, every well got 100µl containing 10000 cells?
Use the formulas: C=n/v (concentration = number you want divided by volume). In your case volume =0.1 ml (100 ul) and n=10,000, so C would = 100,000 cells/ml. The total number of cells you need will be 700,000 cells (it pays to account for pipetting error in your calculations, so you may want more cells.
Assuming that you know how to use a haemocytometer, you should have a number of cells per ml in a certain volume. Rearrange the above equation to give you n (in this case total number of cells) will tell you if you have enough cells or not.
If you do have enough cells, either resuspend them at the desired concentration, or resuspend them so that you can easily take the desired amount into a new tube and dilute. For example if you have 10 million cells total (1x10^7 cells), this is far too many to resuspend at 100,000/ml (v=n/c =100 ml), so you could resuspend in 10 ml to give you 1 million cells/ml and take 0.7 ml to a new tube with 6.3 ml medium give you a 700,000 cells total in 7 ml.
Thank you so much..i understood that..
I use countess chamber from invitrogen..sometimes use hemacytometer..
ok, let say i take 1 flask , then when i calculate, i dont have enough cells.
i take another flask and count
how i going to added both so that i get the desired volume of cells.
can you explain and give example...
really appreciated your words..thank you
The easiest way would be to mix the two flasks' contents and re-count...