3 replies to this topic

[bio08]

Hello,
My friend have Cellsuspension in 5ml medium. The cells got diluted 1:20 and counted the cells.
125 cells* 20 (dilution)*10,000=250*10^6cells/ml

250*10^6cells*5ml= 125*10^6 cells total
The cells centrifuged and the pellet diluted in 1ml

The desired cellamount is 1miljon cells /ml, but the cells will be seeded in  the volume10 ml (and not 1ml).
The cells 125*10^6 cells are now as mentioned diluted in 1 ml medium. So, took 80 ul of this cell suspension and seeded it in 10 ml medium.
Is it right that 80 ul was seeded in 10 ml?
If not, which amount should be seeded in 10ml medium.
Thanks!

[bob1]

Looks about right.  Personally I would have resuspended the cells in a bigger volume than 1 ml for this, as it makes the dilution a bit more accurate.

[almost a doctor]

bio08, on Sep 20 2009, 08:21 PM, said:

Hello,
My friend have Cellsuspension in 5ml medium. The cells got diluted 1:20 and counted the cells.
125 cells* 20 (dilution)*10,000=250*10^6cells/ml

250*10^6cells*5ml= 125*10^6 cells total
The cells centrifuged and the pellet diluted in 1ml

The desired cellamount is 1miljon cells /ml, but the cells will be seeded in  the volume10 ml (and not 1ml).
The cells 125*10^6 cells are now as mentioned diluted in 1 ml medium. So, took 80 ul of this cell suspension and seeded it in 10 ml medium.
Is it right that 80 ul was seeded in 10 ml?
If not, which amount should be seeded in 10ml medium.
Thanks!

If you want 1 million (1*10^6) cells in the 10ml, you actually need 8ul from your dilution.
Although your final cell number is right, your calculations have an error:  

125 cells * 20 * 10,000 = 250*10^5  (or 25*10^6) cells/ml => in n5ml =  25*10^6 x 5 = 125*10^6 cells (or 1.25*10^8)

Now, if you have spun down this cells, and resuspend in 1 ml, you now have 1.25*10^8 cell/ml  if you need 1million cells (whatever the volume you are going to seed them in) :   1*10^6 / 1.25*10^8 = 0.008ml (or 8ul)
If on the contrary, what you need is 10ml at 1*10^6 cells/ml =>  1*10^6 x 10ml / 1.25*10^8 = 0.08ml (or 80ul)

Finally, I agree with bob1, I would have resuspended the cells in a bigger volume.  In fact you don't even need to resuspend your cells (unless you are wanting to change the media),  40ul of your initial 5ml will give you 1million cells, just add this to your 10ml of fresh media, or 9.960ml to be exact.    

Hope this helps.

[bob1]

oops, didn't actually check the initial calculation, just the end bit.