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
