[Cytometry] Scaling problem with FACSDiva

Ed Podniesinski flow8775 at roadrunner.com
Sat Feb 1 09:34:10 EST 2014


Martina,

Have you considered trying a neutral density filter sandwiched on top of
your R670 bandpass filter to cut down the detected photons?
Can't remember where I got my 1" round ND filter kit from ( not at work
right now)
but I have fractional values from .5 to 3.0. Could have been from Thor Labs
or Edmunds.

Ed Podniesinski

Roswell Park Cancer Institute

http://rpciflow.org


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Martina Beyrau
Sent: Wednesday, January 29, 2014 5:36 AM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] Scaling problem with FACSDiva

Dear all,

I would really appreciate some help on the following problem.

When measuring an APC labelled antibody (to a surface protein) on murine
neutrophils using the R670 channel on a Fortessa flow cytometer and FACSDiva
software, both the isotype control and unstimulated labelled cells are set
to have a median fluorescence of approx 300.  Stimulated cells have a
substantially elevated signal, and the range of intensities covers the whole
log scale. In order to fit in the high intensity events the negative values
are shifted to below zero, rather than being in line with the negative
values of the isotype or unstimulated group. This prevents direct comparison
of mean/median fluorescence intensities, or quantifying the % of stimulated
cells which have a higher value than the unstimulated sample.

Can we reset the software so that the low values are aligned, and the high
values will be pushed up against the right hand axis if they exceed the
available log scaling? Or increase the available log scaling so that the
whole range of measurements will fit? I don't want to reduce the voltage too
much as I need to keep the isotype value above zero.

Thanks very much,
Martina

--
Dr Martina Beyrau
Post Doctoral Research Assistant
Queen Mary, University of London
E-mail:    m.beyrau at qmul.ac.uk





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