Re: RE : autofluorescence and compensation

Dear Andrew,

I have heard of dedicating the FITC (FL-1) channel on the Calibur for 
autofluorescence, but not two channels.  Do you know of any example FACs 
plots or can you point me to a paper/ website describing this technique 
for those of us unfamiliar with it?

Thank you,
Kendra

Kendra Hyland, Ph.D.
Discovery Genomics, Inc.
kendrah@discoverygenomics.net




Andrew MITCHELL wrote:
> Hi Eric,
>
> The simplistic take on it is that compensation is solely dependent on the percentage of
> spillover of each fluorochrome between channels, and autofluorescence has no bearing on
> this. Having said that, and it sounds like you know, for initial compensation setup you
> need to have stained and unstained particles that have identical autoflourescence (see
> Mario Roederer's compensation site for a good review). Once this is done however, the
> compensation matrix will be valid for particles of all autofluorescence levels...just
be
> aware that the unstained cells of one type are likely to be in a different position on
a
> plot to unstained cells of another type. Even if you normally do your compensation
using
> cells, BDs comp beads are worth their weight in gold for setting up compensation in
> populations with heterogeneous AF. If you are working in the mouse, set your viability
> stain up on normal mouse spleen and try to only get lymphocytes (live and dead) in the
> scatter gate as they have relatively homogeneous AF.
>
> If you have enough free channels in your experiment, think about dedicating one or more
> solely to autofluorescence. Instead of it being a hindrance it can actually be useful
in
> distinguishing different cell populations, particularly in teasing apart different
> myeloid lineages. The FITC channel on the calibur is good for this because it can be
used
> essentially without any compensation most of the time, but you can use other channels
if
> you compensate the true fluorochromes out using your preferred flavour of
> post-acquisition analysis software (Diva is a pain for this but with FlowJo it can be
> done relatively easily). If you can dedicate 2 (or more) channels to AF, particularly
> with different excitation lasers (though the red laser on the calibur isn't great), you
> can readily distinguish the AF+ cells since AF is typically excited over a broad range
of
> wavelengths and emission generally "correlates" on a bivariate plot to form a diagonal
> "smear" in the 2 channels...though unfortunately you can't really use AF as a
> "fluorescence parameter" in its own right, probably because it's a result of more than
> one fluorochrome species with similar, but non identical spectral properties.
>
> One last tip...when analysing your data it can be best to initially segregate cells on
AF
> level, if you are using a standard hierarchical gating	strategy instead of
cluster
> analysis etc, and only subsequently look at marker expression on cells with similar AF
> levels.
>
> Good luck!
>
> Andrew
>
> Andrew Mitchell Ph.D
> Equipe Giovanna Chimini
> ABCA1 transporters
> Centre d’Immunology de Marseille-Luminy
> CNRS-INSERM-Université de la Méditerranée
> Campus de Luminy, Case 906
> 13002 Marseille, Cedex 9
> France
> Tel:	+33 (0) 4 91 26 94 90
> Fax: +33 (0) 4 91 26 94 30
> e-mail: mitchell@ciml.univ-mrs.fr
> ________________________________________
> De : Eric Shaw [eseric47@googlemail.com]
> Date d'envoi : mercredi 11 juin 2008 18:20
> À : Cytometry Mailing List
> Objet : autofluorescence and compensation
>
> Hi Flowers,
> I have a question.
> I have a tissue dissagregation which will have lots of dead cells and various cell
types
> which most likely have different amounts of autofluorescence. I know I need to use
single
> stained controls (unstained, 7AAD, FITC,PE,APC) but wont my compensation be inaccurate
> for FITC/PE/APC since I am compensating for the whole population- live and dead? And
what
> about the different cell types (epi's, tissue macrophages etc) that can give differing
> amounts of autoflorescence? I compensate with my R1 gate  off FSC/SSC and I am using a
> FACS Calibur. All replies will be appreciated.
>
> Thanks,
>
> Eric Shaw
>
>
>
>