passage number - How to maintain low passage number? (Jul/25/2004 )

Hi!!! to all.
I am dealing with the cell based assay. My problem is My cells respond to my assay only between the passage number 20 to 35. Now if I have only one vial of these cells at the passage number 23, How should I make my stock of freeze downs so that they last for 3 to 4 years?

Another question is How do people actually count the passage number? If I split my cells at double than the split ratio recommended by ATCC in a same flask do I need to go ahead by two passage number or only one? Same way if I freeze my cells at a passage number 23 and then revive after few days does the passage nuber increases by one or it remains 23 only?

Please reply early,
Thanks in advance

Now if I have only one vial of these cells at the passage number 23, How should I make my stock of freeze downs so that they last for 3 to 4 years?

[B] If you have it in liquid nitrogen, theoretically it can last indefinitely. To produce more of the cells, u should subculture that one vial and for each subculture freeze it down so you have many back ups for the next few years.


Another question is How do people actually count the passage number? If I split my cells at double than the split ratio recommended by ATCC in a same flask do I need to go ahead by two passage number or only one?

Only one.

Same way if I freeze my cells at a passage number 23 and then revive after few days does the passage nuber increases by one or it remains 23 only?

I'll put 24.

Hi,
basically every time you trypsinise your cells (they are adherent?) you increase the passage number by one. That means if you split them even at 10times the suggested amount it still goes up by one. Also when freezing you would have trypsinised them before storage, therefore even though the revival itself does not add a passage to your count, the number would be 24.