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Current Protocols in Molecular Biology Jul 2015In 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...
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.
Topics: Animals; Cell Cycle; Cytological Techniques; DNA; Flow Cytometry; Humans; Ki-67 Antigen; RNA; Staining and Labeling
PubMed: 26131851
DOI: 10.1002/0471142727.mb2806s111 -
Nature Chemistry Sep 2022The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every super-resolution fluorescence microscopy (nanoscopy)...
The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every super-resolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging super-resolution methods. Photoactivatable dyes offer substantial improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group-free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogues, yielding a family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell labelling for conventional microscopy, as well as the coordinate-stochastic and deterministic nanoscopies STED, PALM and MINFLUX.
Topics: Fluorescent Dyes; Ionophores; Microscopy, Fluorescence; Silicon
PubMed: 35864152
DOI: 10.1038/s41557-022-00995-0 -
Cytometry. Part a : the Journal of the... Jan 2013Hematopoietic stem cells (HSCs) remain the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional... (Review)
Review
Hematopoietic stem cells (HSCs) remain the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in murine bone marrow is about 0.01% of total nucleated cells and ∼5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. In this updated version of our review from 2009, we review different strategies for hematopoietic stem and progenitor cell identification and purification. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of updating this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.
Topics: Animals; Bone Marrow Cells; Cell Cycle; Cell Proliferation; Coloring Agents; Flow Cytometry; Hematopoietic Stem Cells; Humans; Mice
PubMed: 22736515
DOI: 10.1002/cyto.a.22093 -
Pharmaceuticals (Basel, Switzerland) Mar 2023Tracers for bimodal optical imaging and positron emission tomography unite multiple advantages in a single molecule. Their tumor-specific uptake can be visualized after...
Tracers for bimodal optical imaging and positron emission tomography unite multiple advantages in a single molecule. Their tumor-specific uptake can be visualized after their PET activation by radiofluorination via PET/CT or PET/MRI allowing for staging or therapy planning, while their non-radioactive moiety additionally facilitates the visualization of malignant tissue during intraoperative fluorescence-guided surgery or in histological assessments. The silicon-bridged xanthene core offers the opportunity for radiofluorination with SiFA isotope exchange to obtain a small-molecule, PET-activatable NIR dye that can be linked to different target vectors. Herein, we demonstrate for the first time the PET-activation of a fluorinated silicon pyronine, belonging to a class of low-molecular-weight fluorescence dyes with a large Stokes shift (up to 129 nm) and solvent-dependent NIR dye properties, with a successful radiochemical conversion of 70%. The non-fluorinated pyronine precursor is easily accessible by a three-step sequence from commercially starting material with a 12% overall yield. Moreover, a library of seven unusually functionalized (by approximately 15 nm), red-shifted silicon rhodamines were synthesized in three- to four-step sequences and the optical properties of the novel dyes were characterized. It was also shown that the synthesized silicon rhodamine dyes can be easily conjugated by amide bond formation or 'click-reaction' approaches.
PubMed: 36986500
DOI: 10.3390/ph16030401 -
Animals : An Open Access Journal From... Mar 2022In this study, we present first data concerning the morphological observations of the orbital region, eye tunics, upper and lower eyelids, superficial gland of the third...
In this study, we present first data concerning the morphological observations of the orbital region, eye tunics, upper and lower eyelids, superficial gland of the third eyelid with the third eyelid, and lacrimal gland in captive adult male Asiatic black bear. The following research methods were used in the work: the eyeball morphometry, the orbital region description, macroscopic description, morphometric and histological analysis of the eye tunics and selected the accessory organs of the eye (Fontana-Masson, hematoxylin & eosin (H&E), Methyl-green-pyronin Y (MGP Y), Movat pentachrome, and picro-Mallory trichrome) as well as histochemical examination (PAS, AB pH 1.0, AB pH 2.5, AB pH 2.5/PAS and HDI). The eyeball of the Asiatic black bear was a spherical shape, while the periorbita was funnel/conical-shaped and the eye socket was of the open type. The cornea was absent of the Bowman's membrane similar to all domestic dogs and some wild dogs. There were palisades of Vogt in the corneal limbus epithelium similar to the Canidae. Degenerative choroidal tapetum lucidum similar to ranch mink (Mustelidae) has been found. The pupil was big and round in shape. The ciliary muscle, dilatator and sphincter muscle were well developed, similar to the pinnipeds. The lens was biconvex round, similar to the Canidae. The retina was composed similarly to the diurnal terrestrial carnivores. In both eyelids were observed very well-developed tarsal glands, ciliary glands and sebaceous glands. The orbital zone in the eyelids was characterized by lymphoid follicles, diffuse lymphocytes and specialized high endothelial venules. In the anterior palpebral margin of the upper eyelid, soft and short eyelashes were observed, while in the lower eyelids they were absent. The third eyelid was T-shaped and composed of the hyaline tissue, and it contained CALT, similar to that in Canidae. The superficial gland of the third eyelid was a multilobar alveolar branched complex with seromucous nature, while the lacrimal gland was also a multilobar acinar branched complex gland, but producing a mucous-serous secretion. The results of our research indicate that the features of the anatomy of the eye and orbital region in Asiatic black bear are also typical of the Ursidae family. Moreover, a detailed analysis of the morphological eye region may be useful in comparative studies and veterinary diagnostics in this bear species.
PubMed: 35405790
DOI: 10.3390/ani12070801 -
Cytometry. Part a : the Journal of the... Jan 2009Hematopoietic stem cells (HSCs) remain by far the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess... (Review)
Review
Hematopoietic stem cells (HSCs) remain by far the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains difficult because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in bone marrow is about 0.01% of total nucleated cells and approximately 5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. This prohibits the study of processes in HSCs, which require large amounts of starting material. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. This report will review different hematopoietic stem and progenitor purification strategies and compare flow cytometry profiles for HSC sorting and analysis on different instruments. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.
Topics: Animals; Biomarkers; Bone Marrow Cells; Cell Separation; Flow Cytometry; Hematopoietic Stem Cells; Humans; Mice
PubMed: 19023891
DOI: 10.1002/cyto.a.20674 -
Veterinary Pathology Mar 2009Utilization of a combined Alcian Blue and Pyronine Y histochemical method for the assessment of multiple parameters in the respiratory tract of various species is...
Utilization of a combined Alcian Blue and Pyronine Y histochemical method for the assessment of multiple parameters in the respiratory tract of various species is described. Acidic mucins were deep blue (sialylated mucins), red (sulfated mucins), or variably purple (mixture of sialylated/sulfated mucins), and differential mucus production was readily detected in a murine respiratory syncytial virus vaccine model of pulmonary inflammation. Elastic fibers stained red in the walls of pulmonary arteries, connecting airways, alveolar septa, and subpleural interstitium. Mast cells had red to red-purple granular cytoplasmic staining. Nuclei were ubiquitously counterstained pale blue. Representative staining was detected in tissues from multiple species, including inbred mice, rats, ferrets, cats, dogs, sheep, and pigs. The fluorescent property of the stained tissues offers additional modalities with which to analyze tissue sections. This histochemical technique detects multiple critical parameters in routine paraffin sections of lung tissue, reduces the need for repeated serial sectioning and staining, and is cost-effective and simple to perform.
Topics: Alcian Blue; Animals; Carnivora; Disease Models, Animal; Lung Diseases; Mice; Pyronine; Rabbits; Rats; Respiratory System; Sheep; Staining and Labeling; Swine
PubMed: 19261646
DOI: 10.1354/vp.46-2-325 -
Iranian Journal of Pathology 2020Cell population and turnover are controlled by a balance between cell proliferation and apoptosis. Detection of apoptosis in oral cancer contributes to its better...
BACKGROUND & OBJECTIVE
Cell population and turnover are controlled by a balance between cell proliferation and apoptosis. Detection of apoptosis in oral cancer contributes to its better prognosis and improved management. This study aimed to quantify apoptotic cells in leukoplakia and oral squamous cell carcinoma (OSCC) using methyl green-pyronin (MGP) and hematoxylin and eosin (H & E) staining.
METHODS
The sample included a total of 130 subjects (comprising 108 males and 22 females). Formalin fixed and paraffin embedded tissues were used and categorized into three groups of normal oral mucosa (n=10), leukoplakia with dysplasia (n=60), and OSCC (n=60). The number of apoptotic cells and apoptotic index (AI) were calculated after staining with MGP and routine H & E stained slides.
RESULTS
MGP stained the condensed chromatin of apoptotic cells. Statistically significant difference (≤0.001) was observed among various study groups in terms of numbers of AI and apoptotic cells. Also, AI increased with increasing grades of dysplasia, and it was the highest in well differentiated OSCC. Results were statistically significant in both H & E and MGP stained sections (≤0.001). A good correlation was found between MGP and H & E staining results.
CONCLUSION
MGP is more specific and can lead to intense staining for chromatin in apoptotic cells. Accordingly, it can provide a good alternative to H&E in identifying apoptotic cells.
PubMed: 32754214
DOI: 10.30699/ijp.2020.107263.2115 -
Nature Communications Jul 2021Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex...
Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex biological processes and interactions. Vibrational microscopy has great potential owing to the sharp linewidth of vibrational spectra. In 2017, we demonstrated the coupling between electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy with a proposed library of 9-cyanopyronin-based dyes, named Manhattan Raman Scattering (MARS). Herein, we develop robust synthetic methodology to build MARS probes with different core atoms, expansion ring numbers, and stable isotope substitutions. We discover a predictive model to correlate their vibrational frequencies with structures, which guides rational design of MARS dyes with desirable Raman shifts. An expanded library of MARS probes with diverse functionalities is constructed. When coupled with epr-SRS microscopy, these MARS probes allow us to demonstrate not only many versatile labeling modalities but also increased multiplexing capacity. Hence, this work opens up next-generation vibrational imaging with greater utilities.
Topics: Coloring Agents; HeLa Cells; Humans; Models, Chemical; Molecular Probes; Molecular Structure; Nonlinear Optical Microscopy; Optical Imaging; Pyronine; Spectrum Analysis, Raman; Vibration
PubMed: 34312393
DOI: 10.1038/s41467-021-24855-6 -
Biomolecules Sep 2016There is a need to identify novel scaffolds and targets to develop new antibiotics. Methylene blue is a phenothiazine derivative, and it has been shown to possess...
There is a need to identify novel scaffolds and targets to develop new antibiotics. Methylene blue is a phenothiazine derivative, and it has been shown to possess anti-malarial and anti-trypanosomal activities. Here, we show that different phenothiazine derivatives and pyronine G inhibited the activities of three structurally different bacterial RNase P RNAs (RPRs), including that from Mycobacterium tuberculosis, with Ki values in the lower μM range. Interestingly, three antipsychotic phenothiazines (chlorpromazine, thioridazine, and trifluoperazine), which are known to have antibacterial activities, also inhibited the activity of bacterial RPRs, albeit with higher Ki values than methylene blue. Phenothiazines also affected lead(II)-induced cleavage of bacterial RPR and inhibited yeast tRNA(Phe), indicating binding of these drugs to functionally important regions. Collectively, our findings provide the first experimental data showing that long, noncoding RNAs could be targeted by different phenothiazine derivatives.
Topics: Anti-Bacterial Agents; Antipsychotic Agents; Bacterial Proteins; Lead; Phenothiazines; RNA, Bacterial; RNA, Fungal; RNA, Transfer; Ribonuclease P
PubMed: 27618117
DOI: 10.3390/biom6030038