splitting cells

[Ferland Louis H.]

On Thu, 24 Oct 1996, Edmundo Castro wrote:

> Date: Thu, 24 Oct 1996 14:55:57 -0700
> From: Edmundo Castro <ecastro at physci.ucla.edu>
> To: methods at net.bio.net, RUTH MCMAHON <RMCMAHON at ollamh.ucd.ie>
> Subject: Re: splitting cells
> 
> Greetings,
> 
> I believe that the reason why you want to rinse the cells prior to the 
addition of the 
> trypsin is due to the inhibition of trypsin by serum (Ca++, other 
stuff?).  


When I was a student, I was taught the main factor here is that serum is 
quite rich in protein and quenches the trypsin. About Ca++, I don't know 
whether it inhibits trypsin, but I do know that it is required for the 
integrity of desmosomes, structures involved in cell-cell cohesion. That 
is the reason I heard for the use of trypsin-EDTA (or Versen) rather than 
trypsin alone (both prepared in PBS). Not all cell types form desmosomes, 
though, and even those that do can often be trypsinized without EDTA; 
only, they'll come off as a sheet of cells that will require some 
pipetting up and down to free isolated cells (some cells do not like 
this, so use of EDTA is really recommended in these cases).

My $0.02's worth.

- Louis

If your cells 
> are kept with media and serum then you should wash them.  If in serum 
free than it is 
> OK to trypsinize without washing.  Even if you had the cells with 
serum if you are 
> adding an excess of trypsin you may be able to digest enough to 
separate the cells. > 
>  In our lab we use a Puck's-EDTA solution to rinse just to be sure and 
then we > trypsinize.


We just rinse with a few milliliters of PBS (sterile, of course!). It is 
quite cheap and has been working perfectly well for me, with a variety of 
cell lines and types over more than 10 years.

Another $0.02's worth. That's $0.04's so far; I'll send you the bill 
later ;)}

- Louis

> 
> If anyone has other opinions or comments please let me know,  thanks.
> 
> E. Castro-Vargas
> 


Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757     FAX: (514) 843-2719