Schock freezing - cell disruption? - (Oct/23/2007 )

Once more.
I use adherent cells. In some of my experiments I wash cells with PBS and put the plate with cells directly in -80ºC freezer and when I need them I just take them out and scrap them off. . .
Does this fast (shock) freezing (-80ºC) cells lead to their disruption and/or even cell organelle disruption?

Thanks.
vic

-victor.m-

QUOTE (victor.m @ Oct 23 2007, 07:19 AM)

as lysosomes are disrupted you may get massive problems with proteolysis; use more protease inhibitors than normally

-The Bearer-

QUOTE (The Bearer @ Oct 23 2007, 06:59 AM)

QUOTE (victor.m @ Oct 23 2007, 07:19 AM)

as lysosomes are disrupted you may get massive problems with proteolysis; use more protease inhibitors than normally

Thanks Bearer for suggestion.
But then I would have a question, if I would not quickly freeze the cells (as I described) and I would do direct (after washing cells with PBS) scraping them on ice and/or in cool room, cells and possibly lysosomes could also be mechanically disrupted (by scraper) or not?
Other point is that I want to avoid using trypsin to detach cells because my protein should be plasma membrane spanning protein and trypsin could digest it.
v.

-victor.m-

QUOTE (victor.m @ Oct 23 2007, 11:05 AM)

QUOTE (The Bearer @ Oct 23 2007, 06:59 AM)

QUOTE (victor.m @ Oct 23 2007, 07:19 AM)

as lysosomes are disrupted you may get massive problems with proteolysis; use more protease inhibitors than normally

it is a question of time: thawing shock-freezed cells give proteases time to work; rapid lysis, dilution of lysate and usage proteases inhibitors should be quicker; nevertehless you may try show-freeze whole cells but it is not recommended

-The Bearer-