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Autofluorescence and Apoptosis in Flow Cytometry: Correlative Methods for Fluorescence and Confocal Microscopy

Published online by Cambridge University Press:  02 July 2020

R. W. Smith
R. W. Smith*
Affiliation:
Environmental Sciences and Resources Program-Biology, Portland State University, P.O. Box 751, Portland, Oregon , 97207 Oregon Health Sciences University, Department of Pathology, L-471, 3181 SW Sam Jackson Park Road, Portland, Oregon , 97201
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Abstract

Flow cytometry offers several advantages in evaluating cellular fluorescence and separating values from chemical probes or transfected fluorescent proteins from autofluorescence. Autofluorescence can introduce artificial values that cannot be separated by examination of the fluorescence histogram. Often a comparison of fluorescence in two detectors can separate autofluorescence from positive fluorescence. A region can be defined around the cells of interest for further statistical evaluation or for subsequent sorting.

Autofluorescence is also correlated with apoptosis in that apoptotic cells often increase autofluorescence values. A clear separation of living cells from dying cells can be achieved by the introduction of a DNA stain, such as 7AAD, which will penetrate cells with disrupted or damaged cellular membranes. Intact living cells will not show the 7AAD fluorescence.

This separation is important in transfected fluorescent protein studies, as the transfection process is often toxic to cells.

Type
Correlative Fluorescent Microscopy and Flow Cytometry Techniques (Organized by R. Smith)
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 618 - 619
Copyright
Copyright © Microscopy Society of America 2001

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References

1.Shapiro, H. M., Practical Flow Cytometry, Third Ed., New YorkWiley-Liss (1995) 542.Google Scholar
2.Dawn Grant, T., et.al. Developmental Dynamics Wiley-Liss, New York, 218 (2000) 394–400.3.0.CO;2-I>CrossRefGoogle Scholar
3. Acknowledgments: This work possible through the generous encouragement of Dr. David T. Clark at Portland State University and the flow cytometry core facilities at Oregon Health Sciences University, the Oregon Cancer Center and the Veteran&s Administration Medical Center, Portland, Oregon.Google Scholar
4. 7AAD used in this study was supplied by Molecular Probes, Eugene, Oregon.Google Scholar