5 replies to this topic

[Iwenim]

Hello everybody,

I am working with HaCat cells. Since I would like to maintain surface-proteins I would like to avoid trypsin.
Well, therfore I used a cell scraper but after scraping the cells they neither become adherent nor they grow anymore (sure because they are´nt adherent anymore).

Does anybody knows this phenomen or knows what I am destroying by scraping these cells??

THANKS a lot!
Iwenim

[bob1]

The cell scraper will physically damage the cells as they are scraped off the surface... this is why they don't grow so well after scraping.  You could probably use a collaginase or even just EDTA in PBS to lift the cells.

[rhombus]

Iwenim, on 25 March 2011 - 01:47 AM, said:

Hello everybody,

I am working with HaCat cells. Since I would like to maintain surface-proteins I would like to avoid trypsin.
Well, therfore I used a cell scraper but after scraping the cells they neither become adherent nor they grow anymore (sure because they are´nt adherent anymore).

Does anybody knows this phenomen or knows what I am destroying by scraping these cells??

THANKS a lot!
Iwenim

Another tactic would be trying several tissue culture and non tissue culture treated plates/flasks/dishes. All the TC manufacturers have different surface charges and chemical compositions. You can optimise your conditions such that the cells JUST adhere to the TC surface....and like Bob suggest use EDTA/PBS to detach the cells....no need for enzymatic removal.

Scraping is a definite "No No" when trying to subculture cells.

Just a thought.

Kindest regards.

Uncle Rhombus.

[Iwenim]

Hey,
thanks a lot!!

I did it with EDTA and it worked very well. You just have to wash the cells with PBS containing Ca++/Mg++  - than the cells grow nice again.

And different TC-surfaces let them grow differently after the EDTA treatment  - but it is more equal after Ca++/Mg++ washing!


Thanks for all the hints!

[ldoreliasimona]

Hi all,
Could you please let me know what is the EDTA concentration needed for the detachment of the HaCaT cells. I was trying 0.5 mM, and it was without succes.
A second attempt was with 0.25% Trypsin-EDTA solution, 5 minutes at 37C and only few cells were detached.

Thanking you in advance,
Simona

[h9494036]

it is not a good idea to scrap the cells unless you are only collecting proteins from cells.