question about analog compensation using beads

Ian Dimmick 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 !!


Ian


Ian Dimmick
Flow Cytometry Core Facility Manager
North East England Stem Cell Institute
Bioscience Centre
International Centre for life
Newcastle Upon Tyne
NE1 3BZ
UK

Ian.Dimmick at ncl.ac.uk
Tel	 0044 191 2418831
Fax	0044 191 2418666
(mob)  0044 7970344823

-----Original Message-----
From: Nicholas Ostrout [mailto:nicholas.ostrout at case.edu] 
Sent: 07 August 2007 20:34
To: cyto-inbox
Subject: Re: question about analog compensation using beads

Dahui,

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 
there.	

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
PhD Candidate
Center for Global Health and Diseases
Case Western Reserve University School of Medicine
216-368-6257



----- 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
To: cyto-inbox

> 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
> Tel:225-578-5960
> Fax:225-578-2594
> 
> 
> 
> 
>	    
> 
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