calculating the number of cells to freeze down - (Nov/24/2011 )

Dear all,
i desparately need your help with the following.

I need to start growth curve experiments that will run over 7 days, my supervisor wants me to seed 1x10⁵ cells in 20ml of medium in each T75 flask and im struggling to calculate

i need to freeze down SK-N-SH cells. the final concerntration of cells should be 3x10^6. the total number of cells i have in 20ml is 3.14x10⁷.

If I understand your question correctly you have a sample that contains 3.14x10⁷ cells in 20 ml and you need to seed 1x10⁵ cells in fresh 20ml?

If so: if 20ml contains 3.14x10⁷ cells, then how much cells are there in 1ml? And how much you need to take to end up with 1x10⁵ cells ?

3.14x10⁷cells/20ml = 1.570.000cells/ml ==> how much you need to take to have 100.000 ? Cleary 1ml still contains too many cells...

If I were you I'd:

1) Spin down my SK-N-SH cells and resuspend in 1ml of media. that 1ml should contain 3.14x10^7 cells.
2) Aliquot 100 microL into a cyrovial and top-up the desire amount of media and cyro preservatives for freezing.
- This would have ~3 million cells.
3) To get 1x10^5 in 20ml, just use this formula:
Initial concentration(M1) X Initial Volume (V1) = Final Concentration (M2) X Final Volume (V2)
=> Initial Volume (V1) = <0.1 X 20ml>/31.4 = ~0.06369ml or 63.69 microL

Simple calculations hehe