The effect of trypan blue treatment on autofluorescence of fixed cells.

Autor: Shilova ON; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya St., 16/10, Moscow, 117997, Russian Federation., Shilov ES; Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow, 119234., Deyev SM; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya St., 16/10, Moscow, 117997, Russian Federation.; Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow, 119234.
Jazyk: angličtina
Zdroj: Cytometry. Part A : the journal of the International Society for Analytical Cytology [Cytometry A] 2017 Sep; Vol. 91 (9), pp. 917-925. Date of Electronic Publication: 2017 Aug 30.
DOI: 10.1002/cyto.a.23199
Abstrakt: 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.
(© 2017 International Society for Advancement of Cytometry.)
Databáze: MEDLINE
Externí odkaz: