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Circulating cell-free nucleic acids are considered a promising source of biomarkers for diseases and cancer. Liquid biopsy biomarkers for brain tumours represent a major, still unmet, clinical need. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Here we report an easy and reproducible method to analyse cell-free nucleic acids in plasma and EVs by conventional flow cytometry easy to translate into the clinics. Nucleic acids associated with the EVs or present in plasma samples are stained by Pyronin Y, which is a fluorescent dye that is preferably binding double-stranded nucleic acids. Fluorescent staining of EVs isolated from cell-conditioned media is suitable for DNA and RNA detection by flow cytometry. The nucleic acids are partially protected from degradation by the EVs' membrane. Additionally, DNA and RNA can be stained in plasma samples and plasma-derived EVs. Remarkably, analysis of plasma from patients and healthy individuals reveals a difference in their nucleic acid profiles. Taken together, our results indicate that the proposed methodology, which is based on conventional direct flow cytometry, is a promising easy tool for plasma nucleic acid analysis.

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Flow cytometry methods for sorting specific populations of cells based on fluorescence or physical properties have been a widely used technique for decades. Flow cytometry has been particularly vital to the study of planarians, which remain refractory to transgenic transformation, as it has provided a work-around solution for studying stem cell biology and lineage relationships in the context of regeneration. Many flow cytometry applications have been published in planarians, beginning with broad Hoechst-based strategies for isolating cycling stem cells and progressing to more function-based approaches involving vital dyes and surface antibodies. In this protocol, we look to build on the classic DNA-labeling Hoechst staining strategy by adding pyronin Y staining to label RNA. While Hoechst labeling alone allows for the isolation of stem cells in the S/G2/M phases of the cell cycle, heterogeneity within the population of stem cells with 2 C DNA content is not resolved. By considering RNA levels, this protocol can further divide this population of stem cells into two groups: G1 stem cells with relatively high RNA content and a slow-cycling population with low RNA content, which we call RNAlow stem cells. In addition, we provide instruction for combining this RNA/DNA flow cytometry protocol with EdU labeling experiments and describe an optional step for incorporating immunostaining prior to cell sorting (in this case with the pluripotency marker TSPAN-1). This protocol adds a new staining strategy and examples of combinatorial flow cytometry approaches to the repertoire of flow cytometry techniques for studying planarian stem cells.

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Peroxyoxalate chemiluminescence (POCL) systems have received great attention due to their high quantum yield and the ability to emit a wide-range colors by the utilizing different fluorophores. In this research, Pyronin Y (PY) was first introduced as the fluorophore for a POCL system. Our results indicated that the reaction of (2, 4-dinitroophenyl) oxalate (DNPO) with hydrogen peroxide (H2O2) catalyzed by sodium salicylate (SS) could transfer energy to Pyronin Y via the formation of the dioxetanedione intermediate and emit orange-red light. The relationships between chemiluminescence (CL) intensity and the concentrations of DNPO, fluorophore, H2O2, and the catalyst were investigated. Moreover, the analytical utilization of the new CL system was evaluated by detecting a drug, cysteamine, in pharmaceuticals. A linear working range for cysteamine concentrations from 3 × 10 -8 to 7.5 × 10 -6 molL-1 (r > 0.9907, n = 5) and a detection limit of 7.8 × 10-9 molL-1 were obtained, respectively. The relative standard deviation for five repetitive determinations was less than 3.8 %, with estimated recoveries of 100.1 % and 103.4 %. This method shows high sensitivity for the assay of cysteamine.

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In this chapter, four methods are described that can be used to assess cell cycle status in flow cytometry. The first method is based on the simultaneous analysis of cellular DNA content using a fluorescent DNA dye (propidium iodide) and of a nuclear proliferation marker (Ki-67). The second is based on the differential staining of DNA and RNA using Hoechst 33342 and Pyronin Y: this method is particularly useful to distinguish quiescent cells in G0 phase from G1 cells. Finally, two methods are described based on DNA incorporation of the synthetic nucleosides BrdU and EdU.

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BACKGROUND: Oral exfoliative cytology is a noninvasive and nonpainful technique for early diagnosis of oral potentially malignant disorders and oral cancer, and the use of cytomorphometry ameliorates its diagnostic reliability. The objective of the present study was to analyze methyl green-pyronin Y (MGP)-stained oral exfoliated cells (OECs) of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) by cytomorphometry. MATERIALS AND METHOD: An observational study was conducted on 150 individuals equally divided into three groups: normal mucosa, OL, and OSCC. Smears were prepared from OECs and stained with MGP. Cytomorphometry was done for 100 cells per subject, and various cell and nuclear parameters were measured and calculated. RESULTS: The Kruskal-Wallis test with post hoc correlation showed significant differences in nucleus and cell diameter (ND, CD), nucleus and cell area (NA, CA), nucleus and cell perimeter (NP, CP), and nucleus to cytoplasmic (N:C) ratio for diameter, perimeter, and area. Spearman's ρ correlation of various N:C ratio methods showed good correlation between N:C perimeter and diameter ratio, N:C diameter and ellipse ratio, and N:C area and ellipse ratio. Additional morphological factors showed significant relations for both cell and nuclear regularity factor, shape factor, and nuclear contour index. DISCUSSION: MGP-based cytomorphometry showed a significant decrease in CD, CA, and CP and increase in ND, NA, NP, and N:C ratio from normal mucosa to OL and OSCC. MGP proved its worth as an effective stain for OECs, despite its strict standardization.

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Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.

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In this study, photophysics and photodynamical properties of Pyronin Y (PyY) in different liquid media were investigated. Interactions of PyY, which is a positively charged pigment compound pertaining to the xanthene derivatives with surfactants possessing distinct charges, were determined by using the molecular absorption and fluorescence spectroscopy techniques. It was observed that band intensities of absorption and fluorescence spectra belonging to PyY increase in proportion to the water when compared to three micelle systems, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X-100 (TX-100). This suggests that interactions in micelle systems are different from those in deionized water, and solvation and surface interactions modify. It is determined that the strongest interaction occurs between PyY dye and SDS, anionic surfactant, and this interaction arises from the electrostatic forces. Calculated photophysical parameters indicated that the microenvironment of PyY in SDS micelle is different to that of other systems. In temperature studies, it was reported that increasing the temperature of the samples increased non-radiative transitions. Steady-state fluorescence anisotropy values were calculated by using fluorescence intensities of PyY compound in pre-micellar, micellar and post-micellar systems. Once the PyY fluorescence probe is added to the surfactant containing solutions below the critical micelle concentrations, the measured anisotropy values were found to be low because the probe remains in the deionized water phase. When the surfactant concentration of the medium becomes closer to the critical micelle concentrations, the steady-state anisotropy value prominently increases. This is because of the restrictions on the rotational diffusion of the probe in micellar solution. It is observed that positively charged PyY shows a higher affinity to the negatively charged SDS compared with the positively charged CTAB and neutral TX-100 surfactants. This can be explained by Coulombic interactions.

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The photophysical properties and photodynamics of Pyronin Y (PyY) dye compound in seven polar protic solvents (n-alcohols) were examined as a function of temperature by using UV-visible, steady-state and time-resolved fluorescence spectroscopy techniques. To understand dye-solvent interactions, photophysical parameters including Stokes' shifts, fluorescence quantum yields and fluorescence lifetimes were determined. To examine the effect of solvent polarity, the difference between the ground state dipole moment and the excited state dipole moment was determined. For this purpose, the multiple regression analysis and the Kamlet-Taft technique were used. Moreover, photodynamic parameters, rotational relaxation times and steady-state anisotropy were calculated. The result showed that the specific interactions of PyY with the solvent molecules take place through hydrogen bonding. As the hydrocarbon chain of the alcohols gets longer, photophysical parameters diminish, probably because of weaker hydrogen bonding. Furthermore, it was found out that the dipole moment of excited states (µe ) is higher than that of the ground state (µg ). In addition, Brownian motions increased with the increasing temperature that weakened the fluorescence character of PyY. It was also revealed that the rotation of PyY increased with a prolonged hydrocarbon chain of alcohol series, due to the lesser extent of hydrogen bonding.

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Hoechst 33342 and Pyronin Y double staining can be used to measure DNA and RNA content in live cells by flow cytometry. Quiescent cells at G0 phase have the same amount of DNA as cells at G1 phase but lower RNA levels compared to proliferating cells. Therefore, resting cells in G0 phase can be distinguished from proliferating cells in G1, S, and G2 M phases. This chapter describes a protocol for double staining of live cells with Hoechst 33342 and Pyronin Y. Combined with immunophenotyping of intact and live cells Hoechst 33342 and Pyronin Y staining is a powerful noninvasive method for the analysis and isolation of quiescent cells from any defined cell population.

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The interaction of Pyronin Y with human serum albumin (HSA) has been investigated systematically by fluorescence, absorption, fluorescence decay lifetime measurements, FTIR, synchronous fluorescence spectroscopy, and molecular modeling method. The spectroscopic and fluorescence quenching experiments show that Pyronin Y may show a static quenching mechanism with HSA. The specific binding distance of 1.96 nm between HSA and Pyronin Y was obtained via Förster non-radiation energy transfer method. The thermodynamic parameters indicate that the electrostatic interactions play a significant role during the binding process. In addition, synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of HSA were not influenced with the addition of Pyronin Y. The obtained results can be of biological significance in photodynamic therapy.

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A flexible and free-standing graphene-based hybrid paper was successfully fabricated by successive applications of vacuum filtration and electropolymerization. First, a suspension including graphene oxide (GO) and silver nanoparticles (AgNPs) was prepared, and GO/AgNPs paper was obtained by vacuum-filtration of this suspension through a membrane. This GO/AgNPs paper was transformed to rGO/AgNPs paper by using both chemical reduction with HI and thermal annealing procedures. rGO/AgNPs/poly(PyY) hybrid paper electrode was formed by electropolymerization of Pyronin Y (PyY) on rGO/AgNPs paper electrode from a PyY monomer-containing (pH 1.0) solution. Structural, chemical, and morphological characterization of this hybrid paper was carried out by scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, Raman spectroscopy, infrared spectroscopy, UV-vis absorption spectroscopy, four-point probe conductivity measurement, and cyclic voltammetry techniques. Electrooxidation of nitrite on rGO/AgNPs/poly(PyY) hybrid paper electrode has been achieved at 860 mV with a linear range of 0.1-1000 µM, sensitivity of 13.5 µAµM(-1)cm(-2), and a detection limit of 0.012 µM. Amperometry studies have shown that the hybrid paper electrode is suitable for amperometric determination of nitrite in both standard laboratory samples and real samples. Moreover, this paper electrode selectively detects nitrite even in the presence of 100-fold common ions and exhibits an excellent operational stability and good flexibility.

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In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation-specific marker (Ki-67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle distribution (G1, S, or G2/M), respectively. The second is based on differential staining of DNA and RNA through co-staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, two additional methods for cell proliferation assays with recent updates of newly developed fluorophores, which allow multiplex analysis of cell cycle status, cell proliferation, and a gene of interest using flow cytometry, are outlined.

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The incidence of potentially malignant oral pathology such as leukoplakia, oral submucous fibrosis and squamous cell carcinoma has increased in India. We investigated whether cytoplasmic diameter, nuclear diameter and nucleus:cytoplasm ratio in exfoliative cytology are reliable indicators of potentially malignant lesions. We also investigated methyl green-pyronin Y and Feulgen staining as simple time saving and cost effective staining techniques for diagnostic exfoliative cytology. Cell and nuclear diameters of squamous cells of normal buccal mucosa, oral leukoplakia and oral submucous fibrosis were measured using an ocular micrometer disc. The nucleus:cytoplasm ratios in pathological cells were compared to age, sex and site matched controls. We found a significant reduction in the mean cytoplasmic and nuclear diameter in the experimental groups compared to normal controls. Methyl green-pyronin Y stained smears were clearer than Feulgen stained cells. We suggest that a decreased mean cytoplasmic diameter of exfoliated buccal mucosal cells could serve as an early indicator of dysplastic change in lesions that otherwise appear benign. Methyl green-pyronin Y may be useful for identifying premalignant and malignant transformations before a lesion is visible. The simplicity of the technique makes its routine use feasible.

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