BrdU-FITC / PI cell cycle

Michael Ormerod 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.

Regards,

Michael Ormerod,
1 Furze Place,
Furze Hill,
Redhill,
RH1 1ER

Phone: (0)1737 767879
Mobile: (0)7802293242

Web site: http://uk.geocities.com/m.g.ormerod@btinternet.com/
  ----- 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.
  Associate Professor
  Department of Molecular Genetics and Microbiology
  Graduate Program in Immunology/Virology
  University of Massachusetts Medical School
  55 Lake Avenue North
  Worcester, MA 01655-0002




  -----Original Message-----
  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


  Hi
  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

  Thanks, Sana
  _________________________________________________________________

  http://clk.atdmt.com/UKM/go/msnnkmgl0010000007ukm/direct/01/


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