[Cytometry] LSRII APC issues

Marty Bigos bigos at stanford.edu
Sat Feb 28 15:43:44 EST 2009


Chris -

Given what has been said, I would suspect that the problem is that BD forgot to install the bandpass conditioning filter on the output of the red laser. These lasers are nominally 635 nm, so a 660/20 should be able to provide the OD 6 blocking required for good APC measurements. However they also have "sidebands" of low power at longer wavelengths that will get through a 660/20, but not a 670/14. A way to check is to hold the 660/20 in the red laser light and see if it passes anything.

To correct the "sideband" problem an external notch filter is used on the red laser. This sometimes gets left off by BD (bad quality control). Ask your FE to verify that it is on your laser.

When BD replaced a failed red laser on our LSRII several months back, the exact same problem as yours occurred. We used a 670/14 until BD could get the notch filter sent to us.


Marty
Marty Bigos, Director
Stanford Shared FACS Facility

mail:
Stanford Shared FACS Facility
Beckman Center B015
Stanford CA 94305-5364

Phones:
(office) 650-725-8463
(mobile) 415-845-8450

fax:
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On Feb 27, 2009, at 12:15 PM, Ian Dimmick wrote:

I suspect that you have a 640 red laser that is giving a high background in the 660/20 filter, using the second filter (670/14) you are avoiding any small , but significant amount of red laser light entering your detector , remember the filter labels are not based on finite emissions measured , but on half peak full width , this makes a big difference and is the probable reason why the 640 nm laser is creeping in to the lower 650nm end of your 66/20 band pass . 

Ian 

Ian Dimmick
Flow Cytometry Core Facility Manager
Institute of Human Genetics
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
http://www.nesci.ac.uk/facilities/facs/
________________________________________
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] On Behalf Of Christian Willberg [Chris.Willberg at ndm.ox.ac.uk]
Sent: 27 February 2009 18:51
To: Purdue mailiang list
Subject: [Cytometry]  LSRII APC issues

Dear All,

I was hoping somebody could help us with a problem we're having with
our APC PMT on a new LSRII, our local BD engineer is at a loss.
We have been struggling to get good APC stains with antibodieswe know
work very well on other machines. When we checked with BD rainbow
beads we noticed that the APC dim beads where bunching up halfway
along the axis - this wasn't seen on either the Alexa700 PMT or APC-
Cy7 PMT (please see attached PDF). We checked that this wasn't due to
the filter (660/20) using a second 660/20 filter and the same pattern
was seen. However, when we swapped the 660/20 filter for a 670/14 -
the problem was resolved. Ideally we would like to use the 660/20
filters for APC and not the 670/14. Can anyone explain what could be
causing this and how we can fix it?

Many thanks,
Chris Willberg

Chris Willberg
Biomedical Research Centre - Immunology Theme
University of Oxford
Oxford, UK

Tele: 01865 271290
or tele: 01865 222352
e-mail: Chris.Willberg at ndm.ox.ac.uk
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