BrdU-FITC / PI cell cycle
m.g.ormerod at btinternet.com
Mon Jun 23 13:34:50 EDT 2008
RE: BrdU-FITC / PI cell cyclePersonally I seldom have a problem with spectral overlap in a BrdUrd-FITC PI experiment. If you record the PI fluorescence at 695, you will certainly reduce any spill over from FITC into PI. However, that wavelength is sub-optimal for PI, which is best recorded at a lower wavelength such as 620 nm.
I compensation needs to be applied, it can be applied in the usual fashion.
If you download FCSExpress Reader from the denovosoftware Web site, you can see an example at http://publish.denovosoftware.com/downloads/26E0DEC1-8E80-4B93-8A4F-D17BDC8D7E30/BrdUrd_PI.fe_launch.
A compensation has bee applied in software to correct the small overlap from fluorescein into PI. The larger fluorescence of the G2/M cells compared to G1 in the fluorescein channel was probably due to the increased autofluorescence of the larger cells.
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----- Original Message -----
From: Gerstein, Rachel
To: Cytometry Mailing List
Sent: Friday, June 20, 2008 3:27 PM
Subject: RE: BrdU-FITC / PI cell cycle
this may not be the answer you are looking for, but, if you detect PI in "FL3" ie using a std filter for Cy5-PE (like 695/40), there is almost no compensation needed if the other color is FITC
Rachel M. Gerstein, Ph.D.
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
From: Sana Arancibia [mailto:m709790867 at hotmail.com]
Sent: Thu 6/19/2008 10:38 AM
To: cyto-inbox Subject: BrdU-FITC / PI cell cycle
To study cell cycle, I use PI with anti-BrdU FITC
PI is on linear scale and my BrdU-FITC is on log
How do you compensate this type of plot?
Any help/insight will be very much appreciated
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