[Cytometry] Compensation with liver cells

andrewm@med.usyd.edu.au andrewm at med.usyd.edu.au
Thu Jul 9 23:00:12 EDT 2009


Hello Kunal,

What you are probably seeing is cellular autofluorescence. Kupffer  
cells in particular have AF with an extremely broad excitation range  
and large Stokes shifts, and are highly autofluorescent in the  
channels that you mention. Other myeloid cells of the liver also have  
enough autofluorescence to cause problems in these channels. Trust  
your bead derived compensation over anything you do with cells. In  
fact don't even try to do compensation using liver cells as the  
autofluorescence will make it uninterpretable.

If autofluorescence is your problem, there is, unfortunately, no  
single easy way of dealing with it when you come to interpreting data.  
It has been argued that AF can be treated essentially as a fluorescent  
parameter in itself (eg. Alberti, S. Cytometry, 1987. 8:114) and  
compensated out of other channels, but in myeloid cells the AF  
patterns are heterogeneous and overlapping (i.e. due to the presence  
of multiple endogenous fluorophores - NAD(P)H, flavins, lipofuscin  
etc) which makes this difficult/impossible to do in practice.

Probably your only option is simply to be acutely aware of the  
autofluorescent populations. Depending on what your cells of interest  
are, you may be able to identify them using combinations of antibodies  
that give discrete staining patterns. Also, fluorescence-minus-one  
controls are essential in determining staining postivity/negativity of  
autofluorescent cell populations (apart from all the other reasons to  
use FMO controls).

good luck,

Andrew



Quoting "Chaudhary, Kunal" <kchaudhary at ukaachen.de>:

> Dear fellows,
>
> I have a big problem regarding compensation. I did compensation with  
> beads which was accurate but as soon as i tried to compensate with  
> liver cells its shows huge difference. I dont know which  
> compensation set up to take and which not. I isolate the liver cells  
> by perfusion (Collagenase type 1 + Dnase I) and than digestion again  
> with collagenase + DNase I at 37 degree. Finally i measure my cells  
> and i see again wired results. I didnt add any Ab labelled with  
> percpe Cy7 and APC but still i get strong signals. I rechecked the  
> PMT and compensations and all but i still cannot understand from  
> where i get these signals. Please please help me.
>
> Thank you very much in advance,
> With regards,
>
>
> Kunal Chaudhary
> PhD student
> Department of Medicine III
> University Hospital Aachen
> Aachen, GERMANY
> Tel: +49-241-80-89200
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
>

============================================
==    Andrew Mitchell PhD                 ==
==    Molecular Immunopathology Unit      ==
==    University of Sydney                ==
==    Department of Pathology             ==
==    Medical Foundation Building (K25)   ==
==    92 - 94 Parramatta Rd, Camperdown   ==
==    NSW 2042, Australia                 ==
==    email: andrewm at med.uysd.edu.au      ==
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