The effect of trypan blue treatment on autofluorescence of fixed cells

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. © 2017 International Society for Advancement of Cytometry.

Comparison of Polydopamine-Coated Mesoporous Silica Nanorods and Spheres for the Delivery of Hydrophilic and Hydrophobic Anticancer Drugs

“…Thus, the presence of a PDA coating would reduce the fluorescence of the underlying particles, leading to emission of dull-green fluorescence from PDA-modified MSNs. Further, trypan blue treatment was used for quenching the extracellular fluorescence from non-internalized particles for more accurate FACS measurements and trypan blue reduces the fluorescence signal from dull-green emitters [29]. Thus, the fluorescence is semi-quantitative at best, and quantification is further complicated by particle incorporation [30].…”

Genetic timestamping of plasma cells in vivo reveals tissue-specific homeostatic population turnover

“…background cells. If the underlying reason for the false GFP positivity within the GFP low B220 hi population is autofluorescence; a common issue when GFP is weakly expressed because autofluorescence typically displays similar excitation and emission characteristics to GFP (Shapiro, 1995); approaches that quench autofluorescence may reduce or abrogate background (Shilova et al, 2017). Alternatively, the experimenter may design a more stringent gate, possibly increasing specificity and thus reducing contamination as long as the loss of a fraction of lowly expressing GFP cells is acceptable and consideration is taken that such stringency may itself introduce bias in the analysis.…”

The Mechanism of Fluorescence Quenching of Protein Photosensitizers Based on miniSOG During Internalization of the HER2 Receptor

“…In order to verify the activity of the resulting protein photosensitizers, the specificity of binding of the 4D5scFv-miniSOG and DARPin-miniSOG recombinant proteins to the HER2/neu receptor on the surface of human breast adenocarcinoma SK-BR-3 cells overexpressing HER2/neu was measured by flow cytofluorimetry. This procedure allowed for testing the selectivity of binding between the targeting module of DARPin and the receptor, as well as flavoprotein functionality, since the toxic module miniSOG exhibits intrinsic fluorescence and its binding to the cells can be detected directly [11].…”