From: Andrew Wells (adwells@mail.MED.UPENN.EDU)
Date: Wed Dec 10 2003 - 15:36:07 EST
Michael - we use TOPRO-3 as a vital dye at final concentrations between 1 and 5 nanomolar. The 1 millimolar (in DMSO) stock that comes from Molecular Probes lasts over a year at -20. For use, we make up a 5 micromolar solution in PBS (not FACS buffer). We've noticed that this solution lasts for 1-2 months at 4C, so we only make 1 mL or so at a time. This is now your 1000-5000X stock. You can dilute this further in PBS immediately before use, such that you're adding 10-50 microL to ~0.5 mL cell suspension (which can be in FACS buffer). J.C.I. 100,3173, 1997 AW On Friday, November 28, 2003, at 01:52 AM, Michael Swain wrote: > > Dear Flowers, > Recently we upgraded to a Beckman Coulter FC500 with dual laser (488 nm > Argon, 633He-Ne) and are considering using TO-PRO-3 as the viability > stain > (for human cells) and detecting on FL4 (675 nm). > > I am hopeful that some readers to the list have some experience using > TO-PRO-3 as a viablilty stain. From the literature I have read (mainly > for > DNA Analysis) the concentrations of TO-PRO-3 use vary from between 1 > uM to > 16 uM. In addition the product insert indicates the TO-PRO-3 can be > stored > for upto 12 months at -20C. Can this be extended beyond 12 months at > -20c > or by storing at -80C. > > Any comments / suggestions / advice (and possibly procedures) or > references > would be greatfully received. > > Thanks for your time in reading this post :) > > Regards > > Michael Swain > > Cell Therapy & Flow Cytometry Laboratory > Division of Laboratory Services > Women's and Children's Network > Royal Children's Hospital > Flemington Rd, Parkville > Victoria > Australia 3052 > > > Ph: 61-3-9345-5832 > email: michael.swain@wch.org.au > > Andrew D. Wells, Ph.D. Assistant Professor of Pathology and Laboratory Medicine University of Pennsylvania Joseph Stokes, Jr. Research Institute Biesecker Liver Pediatric Disease Center The Children's Hospital of Philadelphia 916 Abramson Research Center 3516 Civic Center Boulevard Philadelphia, PA 19104 phone: (215) 590-8710 fax: (215) 590-7384
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