[Cytometry] Compensation beads with violet antibodies

McClellan, Steve smcclell at ufl.edu
Fri Dec 11 14:52:29 EST 2009

Hi Megan,
Ironic that you posted this today as just yesterday, I was told by my BD rep that they are aware of this problem....no explination as to what is causing the problem, but bottom line, they are recommending that everyone use cells NOT comp beads for the violet reagents.  Hopefully they can figure out the problem and fix it but until then....use real cells.
Kind regards,

Steve McClellan
Senior Biological Scientist
University of Florida
Interdisciplinary Center for Biotechnology Research(ICBR)-
Flow Cytometry Core Laboratories
352-273-8185 (office)
352-273-8070 (fax)
smcclell at biotech.ufl.edu

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Megan L Michalak
Sent: Friday, December 11, 2009 11:25 AM
To: cytometry at flowcyt.cyto.purdue.edu
Subject: [Cytometry] Compensation beads with violet antibodies

We have a number of clients who are doing 7-9 color experiments on our LSR 
II instrument using the violet, red and blue lasers.  We  request that 
they prepare BD comp beads for us to compensate their samples with, we 
have always had a good experience with them and feel that they are more 
accurate than compensation controls with cells.  For the most part the 
beads work great, but we have noticed a large discrepancy when the beads 
are stained with violet fluorochromes (V450, pacific blue, cascade blue 
etc).  The compensation for violet- all other fluorochromes can range from 
3- 50%.  These seem like very high being that this dye is excited by a 
different laser than all others and the excitation for most violet dyes 
aren't excitable by the red laser at all.  When looking at collected 
samples, the events seem to visually over compensated.  The same clients 
on occasion will bring the same experiments with compensation controls 
that are human blood and the compensation for violet- fluorochrome are all 
1% or less which seems to be reasonable.  The collected data generally 
looks better to.  We have clients that use both e bioscience and BD violet 
conjugated fluorochromes and both human and mouse samples we see the same 
phenomenon.  Has anyone else seen this or had this problem?   Any ideas 
why this happens or what we should do.  We don't want to stop using beads 
since many of the markers people are looking for rare and it is hard to 
compensate when the controls have a minimal amount positive.  Thanks for 
your help.

Megan Michalak
Manager- Cell Analysis Facility
University of Nebraska Medical Center
mmichalak at unmc.edu
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Cytometry at lists.purdue.edu

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