Hi Karim, 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 newsletter! 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, Dirk Prof. Dirk Van Bockstaele, PhD Laboratory of Hematology Head Flow Cytometry Antwerp University Hospital Wilrijkstraat 10 B-2650 Edegem Belgium phone 32 3 821 3900, fax 32 3 825 1148 > ---------- > Van: Karim Vermaelen[SMTP:Karim.Vermaelen@rug.ac.be] > Antwoord naar: Karim.Vermaelen@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 > or > 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! > > Karim > Pulmonary Immunobiology > Ghent University Hosp. >
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