Cell passage question - (Apr/15/2008 )

Hello everyone,

I have a question regarding to the number of cell passages.

I usually resuscitate a vial from my working stock (V79 in liquid Nitrogen) and I label that as a 0 passage and start the numbering from there. But I have been told that I need to take into account the passages I used to create the working stock, and perhaps the master stock as well?? that means that I may have 8 passages already!! and I have all my assays' procedures written not to use more than passage 12.

Any advice on that??

Thanks!

I was told that every time I thaw a new vial I should start with passage 0 and throw them when they reach passage 20 (however, this depends on your cell line. If I observe that they start to grow faster than in the beginning, I `don`t do any experiments with them).

In addition I was taught that I should make the freeze stocks when the cells are in early passage (f.exp. until passage 4-5). I always keep some stocks from the very earliest freeze-down. If I need to make more stocks, I use the next early stocks but freeze down the first 2-3 passages.

As I said it also depends on your cell line and how early passage cells you got from the colleauges.

I always labell the vial with the passage number when I freeze down cells and also on the dish when I subculture them.

i made the experience that how this is handled depends on the lab you are working in.

some people will keep the P-count when vials are frozen because they argue that freezing does not change the age of the culture. and others will start with P0 after thawing arguing that only healthy cells survive this procedure and because of this the cell culture is as good as new after you thawed it.

which of these views is right? who knows? both ways work fine in different cell culture labs all over the world.

i dont think "health" has anything to do with this - if you were dealing with unhealthy cells your experiments would be useless in the first place. passage no is more to do with the inherently self destructive nature of life - you divide enough times you risk corrupting your dna - better to be dead than mess up the gene line.

freezing is like sleep - a brief pause - but youre not really any younger the next day

always carry the p no

different labs may do different things - but we all know there are labs out there that tweak the rules (just enough) so they can get results others cant

this is no excuse for you to follow suit

sometimes you just cant freeze cells (eg keritinocytes) you must source fresh tissue and do your exp's as fast as possible.
cell lines have different rules but still have their limits (hence carrying the p no)

which is more important - getting a result or getting the right result

if someone sent me a cell line telling me "not to worry - just start at zero again" i'd know i was dealing with a muppet and avoid them in the future

d