Reducing cellular autofluorescence in flow cytometry: An in situ method
Article first published online: 6 DEC 1998
Copyright © 1997 Wiley-Liss, Inc.
Volume 30, Issue 3, pages 151–156, 15 June 1997
How to Cite
Mosiman, V. L., Patterson, B. K., Canterero, L. and Goolsby, C. L. (1997), Reducing cellular autofluorescence in flow cytometry: An in situ method. Cytometry, 30: 151–156. doi: 10.1002/(SICI)1097-0320(19970615)30:3<151::AID-CYTO6>3.0.CO;2-O
- Issue published online: 26 DEC 2002
- Article first published online: 6 DEC 1998
- Manuscript Accepted: 31 MAR 1997
- Manuscript Received: 30 JAN 1996
- flow cytometry;
- cellular autofluorescence;
- trypan blue
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. Cytometry 30:151–156, 1997. © 1997 Wiley-Liss, Inc.