MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

Abstract

Background/Aims: MicroRNA (miRNA)-induced suppression of dendritic cells (DCs) has been implicated in many diseases. Therefore, accurate monitoring of miRNA endocytosis by DCs is important for understanding the role of miRNAs in many diseases. Recently, a method for measuring the co-localization of Argonaute 2 (AGO2)-associated miRNAs on laser-scanning confocal microscopy method was proposed to localize the miRNAs. But its definition was limited by the number of observed cells through its accuracy. Methods: In this study, a method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs. miR-590-5p proven to play an important role in tumor immunity. This method enabled the quantification, visualization and localization of the fluorescence intensity in 30,000 individual cells. Results: Using this method, the DCs with different endocytotic ability were distinguished. The behaviour of miR-590-5p during endocytosis under the stimulation of tumor antigen in DCs was observed, binding to its cognate target mRNA and degradation in DCs. Conclusion: This method based on imaging flow cytometry provide an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques.

Open-Source Fluorescence Spectrometer for Noncontact Scientific Research and Education

“…The experiment was conducted using a wired OpenFS with DNA molecules labeled with Cy3 fluorophore. The emission spectra were compared for different concentrations of the Cy3-labeled DNA probe (Figure a), and the spectral shape was in good agreement with the known Cy3 emission spectrum . We compared the performance of OpenFS to measure the Cy3-labeled DNA probe with a conventional fluorometer (Denovix QFX Fluorometer, Denovix Inc.).…”

“…As a model system, we adopted adjacent hybridization probes, which were oligonucleotides labeled with Cy3 fluorophore (550 nm excitation, 565 nm emission) as a donor or Cy5 fluorophore (655 nm excitation, 667 nm emission) as an acceptor. Cy3 and Cy5 are a well-known FRET pair in which the emission spectrum of Cy3 in the range between 530 and 690 nm and the absorption spectrum of Cy5 in the range between 510 and 690 nm overlap . The DNA probes were designed so that Cy3 and Cy5 labels were placed close together on complementary target DNA (synthetic single-stranded oligonucleotide) after hybridization, causing fluorescence energy transfer from Cy3 to Cy5 (Figure a).…”

“…Due to the wide excitation spectra of the dyes, an optimal excitation spectrum range for FRET can be between 440 and 500 nm, and only a part of the spectrum of each LED light source overlaps with the optimal excitation range (Figure S4). 29 Although both LEDs can be adjusted to similar intensity levels after bandpass filtering, the blue LED was used for the following experiments without the risk of Cy5 excitation by accidental light leakage. The blue LED, which is readily available, was used in this work, and their emission wavelength ranges did not perfectly match the excitation wavelength of Cy3.…”

“…The emission spectra were compared for different concentrations of the Cy3-labeled DNA probe (Figure 2a), and the spectral shape was in good agreement with the known Cy3 emission spectrum. 29 We compared the performance of OpenFS to measure the Cy3labeled DNA probe with a conventional fluorometer (Denovix QFX Fluorometer, Denovix Inc.). Figure 2b,c indicates the fluorescence peak intensity of Cy3 versus the concentration of Cy3-labeled DNA measured using the wired OpenFS and the conventional fluorometer, respectively.…”

“…Cy3 and Cy5 are a well-known FRET pair in which the emission spectrum of Cy3 in the range between 530 and 690 nm and the absorption spectrum of Cy5 in the range between 510 and 690 nm overlap. 29 The DNA probes were designed so that Cy3 and Cy5 labels were placed close together on complementary target DNA (synthetic single-stranded oligonucleotide) after hybridization, causing fluorescence energy transfer from Cy3 to Cy5 (Figure 3a). We monitored the change in spectral signals with the concentration of the two probes fixed at 160 nM and the concentration of the target DNA changing from 0 to 160 nM.…”