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Cytometry 1987 Mar;8(2):114-9
A single laser method for subtraction of cell autofluorescence in
flow cytometry.
Alberti S, Parks DR, Herzenberg LA
In flow cytometry cell autofluorescence often interferes with efforts
to measure low levels of bound fluorescent antibody. We have
developed a way to correct for autofluorescence on a cell-by-cell
basis. This results in improved estimates of real staining and better
separation of the fluorescence histograms of stained and non-stained
cells. Using a single laser, two-color fluorescence measurement
system and two-color compensation electronics, autofluorescence and
one fluorescent reagent are measured (rather than two fluorescent
reagents). With fluorescein-conjugated antibodies the signal in the
515 to 555 nm range (green fluorescence) includes both fluorescein
emission and part of the cellular autofluorescence. In the cases we
have investigated, autofluorescence collected at wavelengths above
580 nm ("red") is well correlated with the green autofluorescence of
the cells. A fraction of this red fluorescence is subtracted from the
green fluorescence to produce an adjusted fluorescein output on which
unstained cells have zero average signal. Use of this method
facilitates the selection of rare cells transfected with surface
antigen genes. Culture conditions affect the level of
autofluorescence and the balance between red and green
autofluorescence. When applied with fluorescein-conjugated reagents,
the technique is compatible with the use of propidium iodide for
live/dead cell discrimination.
Cytometry 1997 Jun 15;30(3):151-6
Reducing cellular autofluorescence in flow cytometry: an in situ method.
Mosiman VL, Patterson BK, Canterero L, Goolsby CL
Department of Pathology, Northwestern University, Medical School,
Chicago, IL, USA. VLM646@nwu.edu
Cellular autofluorescence affects the sensitivity of flow cytometric
assays by interfering with detection of low level specific
fluorescence. These detection limits increase with use of protocols,
such as thermocycling and fluorescent in-situ hybridization (FISH),
that can increase intrinsic cellular fluorescence to 5,000-20,000
fluorescein isothiocyanate (FITC) equivalents. In order to improve
signal to noise ratios when using FITC labeled probes in these
procedures, we employed a method using the polyanionic azo dye,
trypan blue, to reduce intracellular autofluorescence. Dyes such as
these are commonly used in immunofluorescent microscopy to reduce
background fluorescence. By using this method, we realized an
approximately 5-fold increase in signal to noise ratio (S/N) in the
direct detection of RNA target probes using flow cytometry. Trypan
blue aided in the resolution of dim surface antibodies, internal
markers and probes, and functions to reduce background
autofluorescence after thermocycling and hybridization. This
technique is rapid and easily applicable for reducing intracellular
autofluorescence, and can be used in single and dual color
applications.
--
Dr. L. VOLKOV
Asst. en Cytometrie -Microscopie
Serv. d'Immunologie Fac. de medecine
3001, 12 av.Nord SHERBROOKE
Quebec, CANADA
J1H 5N4
Tel: (819) 346-1110 ext 4867
Fax: (819) 564-5215
Leonid Volkov <leonid.volkov@chus.qc.ca>
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