On leave from the Istituto Mario Negri, Milano, Italy
A single laser method for subtraction of cell autofluorescence in flow cytometry†
Article first published online: 8 MAR 2005
Copyright © 1987 Wiley-Liss, Inc.
Volume 8, Issue 2, pages 114–119, March 1987
How to Cite
Alberti, S., Parks, D. R. and Herzenberg, L. A. (1987), A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry, 8: 114–119. doi: 10.1002/cyto.990080203
This work was supported in part by National Institutes of Health grants GM-17367 and HD-13025. S.A. was partially supported by the Italian Association for Cancer Research.
- Issue published online: 16 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 30 SEP 1986
- Manuscript Received: 17 JUN 1986
- Antibody detection;
- fluorescence compensation
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, autofluorcscence 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 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.