question about analog compensation using beads
ian.dimmick at newcastle.ac.uk
Wed Aug 8 17:01:57 EDT 2007
Changing FS SS voltages on the PMT's will have no effect on the
compensation at all , obviously you will have to return the voltages to
the appropriate ones for the scatter properties of your cells. It is
sometimes necessary to do this as the compensation beads are quite small
and may well lie below the threshold of the instrument scatter settings
that are appropriate for your cells .
The Key word is optimise everything for your cells , you can change
scatter but do not change any PMT's if they are involved with your
fluorescence detection .
One additional point , remember if you do not see any spectral overlap
over and above your specific fluorescence within a PMT , changing that
PMT voltage upwards may disproportionaly increase small amounts of spill
over to a level to where you will need to compensate , so if you decide
to change 1 PMT, check ALL spectral overlap !!
Flow Cytometry Core Facility Manager
North East England Stem Cell Institute
International Centre for life
Newcastle Upon Tyne
Ian.Dimmick at ncl.ac.uk
Tel 0044 191 2418831
Fax 0044 191 2418666
(mob) 0044 7970344823
From: Nicholas Ostrout [mailto:nicholas.ostrout at case.edu]
Sent: 07 August 2007 20:34
Subject: Re: question about analog compensation using beads
You should not change the voltages on your PMTs, otherwise the
compensation will be wrong. When I use comp particles, you
cannot 'see' them, except at the very top of your FSC/SSC dot plot.
However, if you look at a plot of your colors, you should see them
Therefore, I would optimize on your cells, then just run the particles
through without changing the voltages. When you analyze in FlowJo,
you should be able to compensate just fine. That has been my
experience, but I would be interested to see what others have to say.
As to why voltages influence compensation, I could probably try
and explain it, but I think it is best for someone a little
more experienced to do it. Hope this helps.
Nicholas D. Ostrout
Center for Global Health and Diseases
Case Western Reserve University School of Medicine
----- Original Message -----
From: Dahui You <dahui_you at yahoo.com>
Date: Tuesday, August 7, 2007 2:43 pm
Subject: question about analog compensation using beads
> Hi, all:
> I have a question about the compensation using flowjo after
> acquisition using BD CompBeads.
> When I acquire data on the flow cytometer, I have to change
my SSC PMT
> settings (which has been optimized for my cells) on the beads to get
> the profiles in range. So my questions is when PMT settings changed,
> will it affect the compensation later in FlowJo and why it is or not?
> Thanks a lot.
> I am still confused about the concept behind analog compensation.
> Dahui You
> Department of Biological Sciences
> Louisiana State University
> Baton Rouge, LA 70803
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