[Cytometry] compensation

Ian Titley Ian.Titley at icr.ac.uk
Tue Mar 15 13:25:07 EDT 2011


Thanks Joe - I guess we would assess linearity in practice, preferentially by using software such as CS&T, or manually using beads and ploting PMT values versus say MFI for each detector?

Ian


Ian Titley PhD
Section of Haemato-oncology
Institute of Cancer Research
15 Cotswold Road
Belmont
Sutton
Surrey  SM2 5NG
UK

Tel +44 (0)20 8722 4255



>>> <Joe_Trotter at bd.com> 15/03/2011 17:08 >>>
Ian,

        I think we should refine that, and say that since linearity does 
matter when sampling bright single color controls (as input to a spill 
matrix for compensation), that the positives can be as bright as needed as 
long as they are on-scale within the linear range of the detector.

        Joe


Joseph Trotter
Principal Scientist, BD Fellow / Advanced Technology Group

BD Biosciences
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E-mail: Joe_Trotter at bd.com   Website: www.bd.com 




cytometry-bounces at lists.purdue.edu wrote on 03/15/2011 08:21:55 AM:

> From: "Ian Titley" <Ian.Titley at icr.ac.uk>
> To: "Mario Roederer" <roederer at drmr.com>, "Helen Ferry" 
> <helen.ferry at ndm.ox.ac.uk>, "Ian Dimmick" 
> <ian.dimmick at newcastle.ac.uk>, "Mary A' 'Price" <mprice1 at tulane.edu>
> Cc: "cytometry at lists.purdue.edu" <cytometry at lists.purdue.edu>
> Date: 03/15/2011 08:27 AM
> Subject: Re: [Cytometry] compensation
> Sent by: cytometry-bounces at lists.purdue.edu 
> 
> Absolutely, in fact brighter is better (more photons to play with) 
> as long as it stays on scale!
> 
> Ian
> 
> Ian Titley PhD
> Section of Haemato-oncology
> Institute of Cancer Research
> 15 Cotswold Road
> Belmont
> Sutton
> Surrey  SM2 5NG
> UK
> 
> Tel +44 (0)20 8722 4255
> 
> 
> 
> >>> Ian Dimmick <ian.dimmick at newcastle.ac.uk> 15/03/2011 14:59 >>>
> Incorrect , compensation is not affected by signal intensity , so 
> you are safe 
> 
> 
> Ian
> 
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-
> bounces at lists.purdue.edu] On Behalf Of Price, Mary A
> Sent: 15 March 2011 14:36
> To: Helen Ferry; Mario Roederer
> Cc: cytometry at lists.purdue.edu 
> Subject: Re: [Cytometry] compensation
> 
> Hi Everyone,
> 
> On this thread, I have a question that might be considered basic but
> haven't seen it asked or answered (since it might be very basic and I
> should know it) about the beads and staining. I have yet to use the
> beads or have others to do it since I am concerned that since beads are
> uniform, they will bind the antibody better then a cell will since it
> has more receptors for it to bind to. My concern is that it will
> actually show a brighter signal then what a cell with less binding would
> show therefore throwing off the compensation. Is this not a correct
> assumption? 
> 
> Mary
> 
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu 
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Helen Ferry
> Sent: Tuesday, March 15, 2011 5:02 AM
> To: Mario Roederer
> Cc: cytometry at lists.purdue.edu 
> Subject: Re: [Cytometry] compensation
> 
> Hi 
> 
> Diva does allow you to have a negative population for each compensation
> control. You just need to create a new region for the negative
> population in each of the single-stained controls and delete the
> unstained tube from the compensation matrix. This enables you to have a
> mixture of cells and beads in your compensation controls panel although,
> obviously, you should use either beads or cells for the positive and
> negative in a single control.
> 
> Best wishes
> Helen
> 
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu 
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Mario Roederer
> Sent: 14 March 2011 23:52
> To: cytometry at lists.purdue.edu 
> Subject: Re: [Cytometry] compensation
> 
> Again, I'd like to stress that there are only 3 rules to achieve proper
> compensation:
> 
> For each color in any given panel:
> 
> (1) Your compensation control color must be matched to your experimental
> color
> (2) The positive control must be at least as bright as your experimental
> sample
> (3) The positive control for your compensation sample, when unstained,
> must have the same background fluorescence as the negative control for
> that color.
> 
> That's it. 
> 
> You'll note that there is nothing about PMT voltages, filters,
> autofluorescence, cell type, beads, day of the week, phase of the moon,
> or mood of the operator involved.
> 
> All reasonable problems with compensation can be traced to a violation
> of one of these 3 rules.
> 
> I will note that (current) DiVa software enables a common violation of
> rule #3, by only allowing only one universal negative -- thus preventing
> proper compensation when a mixture of bead-based and cell-based
> compensation controls are used.  Better-designed software does not make
> this fundamental mistake.
> 
> mr
> 
> (PS, I left out one rule: that you use the same instrument fluorescence
> settings for all samples and compensation controls.  Violating this rule
> is more fundamental than anything compensation-related; including it as
> a rule would be akin to specifying that you must turn on the instrument
> before you run any controls.  This would be what I would refer to as an
> "unreasonable problem" with compensation.)
> 
> 
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