[Immunology] Immunophenotyping MSCs

Marcela Fabiana Bolontrade via immuno%40net.bio.net (by MBolontrade from leloir.org.ar)
Wed Jul 2 12:24:39 EST 2008


Hello, 
 
I would appreciate receiving comments about the detachment procedure for
cultured Mesenchymal stem/stromal cells (MSCs) for FACS analysis. 
 
I am starting immunophenotyping these cells and I am concerned about
damaging surface antigens using trypsin; however, trypsin is the only
procedure that is working in my hands right now: 

other methods I tried: 

EDTA and a Cell dissociation buffer (Enzime free, PBS based) from Gibco;
these last two methods were not succesful since the cells formed very
tight clumps which did not come out with filtering methods, and lots of
the cells died.  
 

So I am using trypsin at a low concentration ( 0.05%), and let the cells
stand in medium with serum for one hour to let them recover from the
trypsinization process. 

 

Do you have a detailed procedure for this, with particular emphasis on
(1) trypsin-EDTA concentration (2) time left between trypsinization and
recovery of any damaged surface antigen until FACS analysis. 

 

Any thoughts are welcomed

 

Thank you in advance.

 

Marcela

 

 


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