(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.

• Klíčová slova
• ImageJ, bioimage analysis, genotoxicity, high-throughput, in vitro testing,
• MeSH
• biologické markery analýza   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mikroskopie * MeSH
• pilotní projekty MeSH
• poškození DNA * MeSH
• reprodukovatelnost výsledků MeSH
• valinomycin toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• valinomycin MeSH

Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity, and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type-we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554.

• Klíčová slova
• Cellpose, Fiji, ImageJ, automated, blinded, cells, data analysis, microscopy, unbiased, yeast,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Melting snow fields are an extremophilic habitat dominated by closely related Chlamydomonadaceae (Chlorophyta). Microscopy-based classification of these cryophilic microalgae is challenging and may not reveal the true diversity. High-throughput sequencing (HTS) allows for a more comprehensive evaluation of the community. However, HTS approaches have been rarely used in such ecosystems and the output of their application has not been evaluated. Furthermore, there is no consensus on the choice for a suitable DNA marker or data processing workflow. We found that the correct placement of taxonomic strings onto OTUs strongly depends on the quality of the reference databases. We improved the assignments of the HST data by generating additional reference sequences of the locally abundant taxa, guided by light microscopy. Furthermore, a manual inspection of all automated OTU assignments, oligotyping of the most abundant 18S OTUs, as well as ITS2 secondary structure analyses were necessary for accurate species assignments. Moreover, the sole use of one marker can cause misleading results, either because of insufficient variability within the locus (18S) or the scarcity of reference sequences (ITS2). Our evaluation reveals that HTS output needs to be thoroughly checked when the studied habitats or organisms are poorly represented in publicly available databases. We recommend an optimized workflow for an improved biodiversity evaluation of not only snow algal communities, but generally 'exotic' ecosystems where similar problems arise. A consistent sampling strategy, two- molecular marker approach, light microscopy-based guidance, generation of appropriate reference sequences and final manual verification of all taxonomic assignments are highly recommended.

• Klíčová slova
• 18S rDNA, ITS2 rDNA, Illumina, OTU clustering, Sanger, high-throughput sequencing, oligotyping, red snow, secondary structure, snow algae,
• Publikační typ
• časopisecké články MeSH

In the past decade, automated microscopy has become an important tool for the drug discovery and development process. The establishment of imaging modalities as screening tools depended on technological breakthroughs in the domain of automated microscopy and automated image analysis. These types of assays are often referred to as high content screening or high content analysis (HCS/HCA). The driving force to adopt imaging for drug development is the quantity and quality of cellular information that can be collected and the enhanced physiological relevance of cellular screening compared to biochemical screening. Most imaging in drug development is performed on fixed cells as this allows uncoupling the preparation of the cells from the acquisition of the images. Live-cell imaging is technically challenging, but is very useful for many aspects of the drug development pipeline such as kinetic studies of compound mode of action or to analyze the motion of cellular components. Most vendors of HCS microscopy systems offer the option of environmental chambers and onboard pipetting on their platforms. This reflects the wish and desire of many customers to have the ability to perform live-cell assays on their HCS automated microscopes. This book chapter summarizes the challenges and advantages of live-cell imaging in drug discovery. Examples of applications are presented and the motivation to perform these assays in kinetic mode is discussed.

• Klíčová slova
• Drug development, Environmental control, Image analysis, Imaging, Kinetic, Live cell,
• MeSH
• buněčné kultury MeSH
• kultivované buňky MeSH
• lidé MeSH
• mikroskopie MeSH
• molekulární zobrazování metody   MeSH
• objevování léků * metody   MeSH
• počítačové zpracování obrazu MeSH
• rychlé screeningové testy * MeSH
• software MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Molecular processes involved in gene expression encompass multitudes of interactions between proteins and nucleic acids. Quantitative description of these interactions is crucial for delineating the mechanisms governing transcription, genome duplication, and translation. Here we describe a detailed protocol for the quantitative analysis of protein-nucleic acid interactions based on protein-induced fluorescence enhancement (PIFE). While PIFE has mainly been used in single-molecule studies, we modified its application for bulk measurement of protein-nucleic acid interactions in microwell plates using standard fluorescent plate readers. The microwell plate PIFE assay (mwPIFE) is simple, does not require laborious protein labeling, and is high throughput. These properties predispose mwPIFE to become a method of choice for routine applications that require multiple parallel measurements such as buffer optimization, competition experiments, or screening chemical libraries for binding modulators.

• Klíčová slova
• Binding assay, Cy3, DNA, Protein, Protein-induced fluorescence enhancement (PIFE), RNA,
• MeSH
• DNA chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• nukleoproteiny chemie   MeSH
• proteiny chemie   MeSH
• RNA chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• nukleoproteiny MeSH
• proteiny MeSH
• RNA MeSH

Stem cell-derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient-specific human pluripotent stem cell (hPSC)-derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation-contraction coupling, which determines important functional properties of the hPSC-derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC-derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation-contraction coupling dynamics of the hPSC-derived CMs.

• Klíčová slova
• atomic force microscopy, cardiomyocytes, differentiation, drug screening, drug toxicity, hESC, hiPSC, high-throughput screening,
• MeSH
• biologické modely MeSH
• buněčná diferenciace MeSH
• fenotyp MeSH
• fluorescenční mikroskopie MeSH
• genotyp MeSH
• kardiomyocyty cytologie   MeSH
• lidé MeSH
• mikroskopie atomárních sil MeSH
• pluripotentní kmenové buňky cytologie   MeSH
• rychlé screeningové testy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

OBJECTIVES: Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. METHODS: Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. RESULTS: Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. CONCLUSIONS: We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.

• Klíčová slova
• colorectal cancer, high-throughput expression profiling, long non-coding RNAs, size exclusion chromatography, small extracellular vesicles, transcriptome,
• MeSH
• extracelulární vezikuly * genetika   metabolismus   MeSH
• gelová chromatografie MeSH
• lidé MeSH
• RNA MeSH
• stanovení celkové genové exprese * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA MeSH

Cyanobacteria are important colonizers of recently deglaciated proglacial soil but an in-depth investigation of cyanobacterial succession following glacier retreat has not yet been carried out. Here, we report on the successional trajectories of cyanobacterial communities in biological soil crusts (BSCs) along a 100-year deglaciation gradient in three glacier forefields in central Svalbard, High Arctic. Distance from the glacier terminus was used as a proxy for soil age (years since deglaciation), and cyanobacterial abundance and community composition were evaluated by epifluorescence microscopy and pyrosequencing of partial 16S rRNA gene sequences, respectively. Succession was characterized by a decrease in phylotype richness and a marked shift in community structure, resulting in a clear separation between early (10-20 years since deglaciation), mid (30-50 years), and late (80-100 years) communities. Changes in cyanobacterial community structure were mainly connected with soil age and associated shifts in soil chemical composition (mainly moisture, SOC, SMN, K, and Na concentrations). Phylotypes associated with early communities were related either to potentially novel lineages (< 97.5% similar to sequences currently available in GenBank) or lineages predominantly restricted to polar and alpine biotopes, suggesting that the initial colonization of proglacial soil is accomplished by cyanobacteria transported from nearby glacial environments. Late communities, on the other hand, included more widely distributed genotypes, which appear to establish only after the microenvironment has been modified by the pioneering taxa.

• Klíčová slova
• Cyanobacteria, Glacier forefield, High Arctic, High-throughput sequencing, Primary succession, Proglacial soil,
• MeSH
• biodiverzita MeSH
• DNA bakterií MeSH
• fylogeneze * MeSH
• genotyp MeSH
• ledový příkrov mikrobiologie   MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• sinice klasifikace   genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Arktida MeSH
• Svalbard MeSH
• Názvy látek
• DNA bakterií MeSH
• půda MeSH
• RNA ribozomální 16S MeSH

Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.

• MeSH
• fluorescenční mikroskopie metody   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• mezerový spoj * MeSH
• mezibuněčná komunikace * MeSH
• molekulární zobrazování metody   MeSH
• počet buněk * MeSH
• počítačové zpracování obrazu metody   MeSH
• viabilita buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

3-dimensional (3D) cell cultures are being increasingly recognized as physiologically more relevant in vitro models than traditional monolayer cultures, because they better mimic in vivo-like microenvironment, cell-cell and cell-extracellular matrix interactions. Nevertheless, the broader use of 3D models might be limited by requirements for special consumables, equipment, or skills for 3D cell cultures, and by their limited throughput and scalability. In this study, we optimized and adapted a commercially available agarose-micromolding technique to produce scaffold-free spheroid cultures. Brightfield microscopy was used for routine nondestructive and noninvasive evaluation of spheroid formation and growth. The workflow is compatible with manual, as well as high speed automated microscopic image acquisition, and it is supplemented with an in-house developed macro 'Spheroid_Finder' for open source software Fiji to facilitate rapid automated image analysis. This protocol was used to characterize and quantify spheroid formation and growth of two different hepatic cell lines, hTERT immortalized, but non-cancerous, adult human liver stem cell line HL1-hT1, and human hepatocellular carcinoma cell line HepG2, as well as their responses to a model antiproliferative and cytotoxic agent, 5-fluorouracil. The complete protocol provides a simple and ready-to-use solution to initiate scaffold-free spheroid cultures in any laboratory with standard equipment for mammalian in vitro cell culture work. Thus, it allows to increase throughput and scale of spheroid culture experiments, which can be greatly utilized in different areas of biomedical, pharmaceutical and toxicological research.

• Klíčová slova
• 3D cell cultures, Multicellular spheroids, hepatotoxicity, in vitro toxicity testing,
• MeSH
• buněčné kultury MeSH
• buněčné sféroidy MeSH
• buňky Hep G2 MeSH
• časové faktory MeSH
• fluoruracil farmakologie   toxicita   MeSH
• hepatocelulární karcinom farmakoterapie   metabolismus   patologie   MeSH
• játra účinky léků   metabolismus   patologie   MeSH
• kmenové buňky účinky léků   metabolismus   patologie   MeSH
• lidé MeSH
• nádory jater farmakoterapie   metabolismus   patologie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové antimetabolity farmakologie   toxicita   MeSH
• průběh práce MeSH
• rychlé screeningové testy * MeSH
• screeningové testy protinádorových léčiv MeSH
• testy toxicity MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluoruracil MeSH
• protinádorové antimetabolity MeSH