autofluorescence & isotype control fluorescence
Van Bockstaele, Dirk
Dirk.Van.Bockstaele at uza.uia.ac.be
Mon Oct 23 04:12:50 EST 2000
Looking at your affiliation I reckon you have to cope with highly
autofluorescent macrophages! I would say yes to your last statement and
thus the highly autofluorescent cells are negative for the marker. If you
have a homogenous population (be it low or high in autofluorescence) the
analysis poses no problems. I think you get into problems when you have a
heterogeneous population in terms of autofluorescence. A real positive cell
(with low autofluorescence) will be lost in the background of the highly
autofluorescent cells. A trick to cope with that is using your compensation
circuitry to correct for autofluorescence: since autofluorescence exhibits
(most of the time) a much broader spectrum, you can collect part of it
through an optical path (say for instance fl2 or fl3) that you don't use for
your markers detection (say fl1) and use this to correct your marker
fluorescence: in that way all autofluorescence ends up in the first decade.
You may look at some relevant literature or phone me: we're only 50 km apart
and I wrote something about it in our local Belgian Cytometry Society
Earl A. Timm et al., Technical Innovation: Amplification and detection of a
Y-chromosome DNA sequence by fluorescence in situ polymerase chain reaction
and flow cytometry using cells in suspension. Cytometry 22: 250-255, 1995.
Alberti S. et al., A single laser method for substraction of cell
autofluorescence in flow cytometry. Cytometry 8: 114-119, 1987.
I hope this helps,
Prof. Dirk Van Bockstaele, PhD
Laboratory of Hematology
Head Flow Cytometry
Antwerp University Hospital
phone 32 3 821 3900, fax 32 3 825 1148
> Van: Karim Vermaelen[SMTP:Karim.Vermaelen at rug.ac.be]
> Antwoord naar: Karim.Vermaelen at rug.ac.be
> Verzonden: vrijdag 20 oktober 2000 10:27
> Aan: Cytometry Mailing List
> Onderwerp: autofluorescence & isotype control fluorescence
> Hi everybody,
> Here's maybe a tricky one:
> Suppose you got highly autofluorescent cells and the isotype control is
> in the 2nd-3rd decade. The same isotype control in low autofluorescent
> cells is under the 1st decade.
> Now suppose the signal for marker X on these high autofluorescent cells
> is also in 2nd-3rd decade, and exactly overlaps the isotype control.
> What's the correct interpretation of these results?
> 1) there's no difference between isotype control and marker X, so the
> cells are negative for marker X
> 2) marker X is in the 2nd-3rd decade, so it's definitely positive
> regardless of isotype control background
> Another way to state the question: are fluorochrome fluorescence (be it
> Ag-specific or aspecific binding) and autofluorescence additive signals
> for the same cell?
> TX a lot for any clue!
> Pulmonary Immunobiology
> Ghent University Hosp.
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