The effect of trypan blue treatment on autofluorescence of fixed cells

Olga N. Shilova, Evgeny S. Shilov, Sergey M. Deyev

    Research output: Contribution to journalEditorialpeer-review

    13 Citations (Scopus)

    Abstract

    Controlling background fluorescence remains an important challenge in flow cytometry, as autofluorescence can interfere with the detection of chromophores. Furthermore, experimental procedures can also affect cellular fluorescence in certain regions of the emission spectrum. In this work, the effects of fixation, permeabilization, and heating on cellular autofluorescence are analyzed in various spectral regions, along with the influence of trypan blue as a quenching dye for these treatments. The impact of these procedures on the staining of SK-BR-3 cells with a dim green fluorophore, a miniSOG (mini Singlet Oxygen Generator) flavoprotein in the form of the recombinant protein DARPin-miniSOG, is also evaluated. The data presented here indicate that fixation of certain types of cells leads to noticeable increase of the autofluorescence. Our results also suggest that trypan blue should be used as an autofluorescence quencher only with bright green emitters since it interferes with the fluorescent signal in a longer-wavelength region of the spectrum and as a result causes reduction of the signal from dim green fluorescent agents.

    Original languageEnglish
    Pages (from-to)917-925
    Number of pages9
    JournalCytometry Part A
    Volume91
    Issue number9
    DOIs
    Publication statusPublished - Sep 2017

    Keywords

    • autofluorescence
    • DARPin
    • flow cytometry
    • miniSOG
    • trypan blue

    ASJC Scopus subject areas

    • Pathology and Forensic Medicine
    • Histology
    • Cell Biology

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