> -----Original Message----- > From: Maris Handley [mailto:Maris_Handley@dfci.harvard.edu] > Sent: Thursday, January 10, 2002 11:13 AM > To: Cytometry Mailing List > Subject: HO33342 into 670/20 filter??? > > I am at a loss to explain why cells that are stained with > Hoechst33342 are increasing > the background in my APC signal...HELP! We were looking at > DNA using HO33342 (excited by > UV, emission through a 450/20 filter) and APC (surface > receptor, using 647nm excitation > and emission through a 670/20 filter). When we put on a sample > that was stained with HO > (APC-), the signal in the APC channel went into the higher > end of the second decade. > Hi Maris, We saw the exact same phenomenon a couple years ago with DAPI, I thin k I posted it to the list. Our setup was an Elite with 3 beams: 488 as upper and 633/UV colinear as lower. After some investigation, it appeared the DAPI simply fluoresced up into the 675nm range. It was so bright we were unable to compensate it out, it swamped our APC positive signal, which we could see clearly when there was no DAPI in the tube. OUr solution was to set the 488 and 633 beams colinear as upper beams and run the UV beam alone as the lower beam. What insturment are you running on? Are your beams colinear or separated in time? Tom **************************************************************************** * Thomas W. Mc Closkey, Ph. D. Director of Flow Cytometry, North Shore University Hospital Assistant Professor of Pediatrics, New York University School of Medicine Boas Marks Biomedical Research Center, 350 Community Drive Manhasset, Long Island, New York 11030 ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866 **************************************************************************** *
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:59:20 EST