re: Hoechst; Viable sorting - cell cycle..question?
Michael Fox (MFox@vines.ColoState.EDU)
Fri, 11 Oct 96 15:02:39 MDT
Jeffrey Faust inquired about methods for using Hoechst 33342 to sort viable
cells. We have done this routinely and it is quite simple. You can grow
cells in culture and add HO342 dye solution to the medium to give a final
concentration of 10 to 20 uM. Incubate them for about 1.5 hr, then
trypsinize and resuspend in PBS on ice and analyze as soon as possible. As
Krishan reported some years ago, some cells actively pump Hoechst dye (and
others) out, resulting in poor quality histograms. Adding 15 uM
trifluoperazine or 100 uM verapamil while incubating the cells with Ho342
will give very good staining with CHO cells and probably others that pump the
dye out. You only need a uv blocking filter to block out scattered uv light
unless you are trying to also do surface markers.
Good luck.
Mike Fox
Colorado State University
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web
http://www.cyto.purdue.edu
, EMAIL
cdrom3@flowcyt.cyto.purdue.edu
