Reducing cellular autofluorescence in flow cytometry: An in situ method

Victoria L. Mosiman*, Bruce K. Patterson, Luis Canterero, Charles L. Goolsby

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

100 Scopus citations

Abstract

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 9-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.

Original languageEnglish (US)
Pages (from-to)151-156
Number of pages6
JournalCommunications in Clinical Cytometry
Volume30
Issue number3
DOIs
StatePublished - Jun 15 1997

Keywords

  • Cellular autofluorescence
  • Flow cytometry
  • Trypan blue

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

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