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Related Experiment Videos

Reducing cellular autofluorescence in flow cytometry: an in situ method

V L Mosiman1, B K Patterson, L Canterero

  • 1Department of Pathology, Northwestern University, Medical School, Chicago, IL, USA. VLM646@nwu.edu

Cytometry
|June 15, 1997
PubMed
Summary

Cellular autofluorescence hinders flow cytometry sensitivity. Trypan blue dye effectively reduces this background noise, improving signal detection for RNA probes and cellular markers in complex assays.

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Area of Science:

  • Cellular and Molecular Biology
  • Biotechnology
  • Immunology

Background:

  • Cellular autofluorescence significantly reduces the sensitivity of flow cytometry assays.
  • Protocols like thermocycling and fluorescent in-situ hybridization (FISH) exacerbate autofluorescence, increasing detection limits.
  • High autofluorescence interferes with the accurate detection of low-level specific fluorescence signals.

Purpose of the Study:

  • To enhance signal-to-noise ratios in flow cytometry when using fluorescein isothiocyanate (FITC) labeled probes.
  • To investigate the efficacy of trypan blue in reducing intracellular autofluorescence in flow cytometry.
  • To improve the detection of dim cellular markers and RNA targets after challenging experimental procedures.

Main Methods:

  • Utilized the polyanionic azo dye, trypan blue, to quench intracellular autofluorescence.

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  • Applied trypan blue in conjunction with flow cytometry protocols involving thermocycling and FISH.
  • Evaluated the impact of trypan blue on signal-to-noise ratios for direct RNA target probe detection.

    • Main Results:

    • Achieved an approximately 5-fold increase in signal-to-noise ratio (S/N) using trypan blue.
    • Trypan blue effectively reduced background autofluorescence after thermocycling and hybridization.
    • Demonstrated improved resolution of dim surface antibodies, internal markers, and probes.

    Conclusions:

    • Trypan blue is a rapid and easily applicable method for reducing intracellular autofluorescence in flow cytometry.
    • This technique enhances the sensitivity and reliability of flow cytometric assays, particularly after procedures that increase cellular fluorescence.
    • The method is versatile and can be employed in both single and dual-color flow cytometry applications.