Graphical Workflow Research Articles

GDSA framework, a computational framework for complex modeling problems in radioactive waste management

This paper details a computational framework to produce automated, graphical workflows, and how this framework can be deployed to support complex modeling problems like those in nuclear engineering. Key benefits of the framework include: automating previously manual workflows; intuitive construction and communication of workflows through a graphical interface; and automated file transfer and handling for workflows deployed across heterogeneous computing resources. This paper demonstrates the framework's application to probabilistic post-closure performance assessment of systems for deep geologic disposal of nuclear waste. However, the framework is a general capability that can help users running a variety of computational studies.

Building Domain-Specific Machine Learning Workflows: A Conceptual Framework for the State-of-the-Practice

Domain experts are increasingly employing machine learning to solve their domain-specific problems. This article presents to software engineering researchers the six key challenges that a domain expert faces in addressing their problem with a computational workflow, and the underlying executable implementation. These challenges arise out of our conceptual framework which presents the “route” of transformations that a domain expert may choose to take while developing their solution. To ground our conceptual framework in the state-of-the-practice, this article discusses a selection of available textual and graphical workflow systems and their support for the transformations described in our framework. Example studies from the literature in various domains are also examined to highlight the tools used by the domain experts as well as a classification of the domain-specificity and machine learning usage of their problem, workflow, and implementation. The state-of-the-practice informs our discussion of the six key challenges, where we identify which challenges and transformations are not sufficiently addressed by available tools. We also suggest possible research directions for software engineering researchers to increase the automation of these tools and disseminate best-practice techniques between software engineering and various scientific domains.

Latest features of the ecosystem management decision support system, version 8.0

The Ecosystem Management Decision Support (EMDS) system is a spatially enabled system for environmental analysis and strategic and tactical planning. EMDS combines various sophisticated analytical tools within a GIS environment. Originally released by the Pacific Northwest Research Station, USDA in 1997, EMDS has been maintained and actively extended since then. Building on its core functionality of logic processing and decision modeling and availability as an ArcMap component, recent advances include more advanced geodatabase processing, integration with open-source GIS platforms, incorporation of two new analytical engines, support for scripting tools, implementation of a graphical workflow environment, advanced tactical planning, portfolio management, and a cloud-based collaboration manager. Because EMDS is a generic solution framework, it can be applied to an extremely broad array of problems at virtually any and all spatial scales. This paper presents an overview of the EMDS technology and describes some of the projects in which it has been used.

Asterics: a simple tool for the ExploRation and Integration of omiCS data

BackgroundThe rapid development of omics acquisition techniques has induced the production of a large volume of heterogeneous and multi-level omics datasets, which require specific and sometimes complex analyses to obtain relevant biological information. Here, we present ASTERICS (version 2.5), a publicly available web interface for the analyses of omics datasets.ResultsASTERICS is designed to make both standard and complex exploratory and integration analysis workflows easily available to biologists and to provide high quality interactive plots. Special care has been taken to provide a comprehensive documentation of the implemented analyses and to guide users toward sound analysis choices regarding some specific omics data. Data and analyses are organized in a comprehensive graphical workflow within ASTERICS workspace to facilitate the understanding of successive data editions and analyses leading to a given result.ConclusionASTERICS provides an easy to use platform for omics data exploration and integration. The modular organization of its open source code makes it easy to incorporate new workflows and analyses by external contributors. ASTERICS is available at https://asterics.miat.inrae.fr and can also be deployed using provided docker images.

TIMCPOT: Tcl/Tk Interface for Measuring Crustal Phase-Onset Time

Abstract In recent years, the seismological community has seen an amazing increase in the amount of seismic arrival time picks with the help of artificial intelligence. But, the accuracy of this data remains a challenge to seismologists. In comparison with automatic picks, manual picks have sufficient reliability and accuracy because manual picks are often used not only as a benchmark to evaluate automatic picking algorithms, but also as training samples to train artificial intelligence models. However, measurement through manual labor is a laborious and time-consuming task. To alleviate the manual labor involved in measuring phase arrival time, we developed a graphical interface named Tcl/Tk Interface for Measuring Crustal Phase-Onset Time (TIMCPOT). The TIMCPOT is mainly equipped with two modules: one is designed for measuring seismic arrival time, and the other is for relocating hypocenter. TIMCPOT simplifies user interaction with seismic data, thus reducing most manual labor. TIMCPOT provides several graphical workflows to carry out detection, identification, revision, and comparison operations on measurement work. The measurement procedure can be operated either manually or by autopicker. For the sake of allowing the user to interact easily with seismic data, TIMCPOT is designed to be executed in a semiautomatic model during several necessary procedures such as visually inspecting results and manually modifying picks. By applying a near real-time displaying technique, TIMCPOT can create a travel time curve figure and comparison chart for manual picks in a timely fashion, which provides a rapid way to estimate data quality. The measurement efficiency and travel time data quality have been significantly improved using this graphical interface. An explicit demonstration was shown to validate the powerful performance mentioned earlier. The primary advantage of TIMCPOT is that it reduces manual labor while retaining valuable manual experience on measuring phase picks. Of course, TIMCPOT has several limitations. However, most limitations will be improved in future development.

Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe.

Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N -glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [ 14 C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms. Graphical abstract: Workflow for the labeling, extraction, separation, and identification of LLO species in S. pombe . (A) Radioactive pulse of S. pombe cells with [ 14 C]-glucose for 15 min at 28 °C. (B) Organic extraction of LLOs from labeled yeasts sequentially using methanol, chloroform, H 2 O, chloroform:methanol:H 2 O (1:1:0.3), 0.02 M HCl (to separate glycans from dolichol), and chloroform:methanol:H 2 O (1:16:16). (C) Preparation of the sample for chromatography on paper: drying by airflow and radioactivity check. (D) Loading of samples in chromatographic paper and descendent chromatography in a glass chamber. The obtained plots (CPM versus running distance) need to be analyzed to identify single glycan species.

Evaluating Human Natural Killer Cells Antibody-dependent Cellular Cytotoxicity (ADCC) Using Plate-bound Anti-CD16 Antibodies.

Natural killer (NK) cells are large granular lymphocytes that keep in check the health of neighboring cells through a large array of intrinsically expressed germline-coded receptors. Most importantly, CD16 is a low affinity Fc receptor for IgG that mediates the antibody-dependent cellular cytotoxicity (ADCC) of NK cells, bridging the innate and adaptive immunities. There has been a significant interest in genetically engineering NK cells to enhance its ADCC, with the ultimate goal to produce off-the-shelf NK cell therapy products that can be combined with target-specific monoclonal antibodies to improve clinical outcomes. Previous protocols of ADCC assays use complex cell-based antigen-antibody models, which are both costly and time-consuming. This current protocol is devoid of target cells and uses plate-bound immobilized anti-CD16 antibodies as the trigger. It greatly shortens the experimental time, while faithfully evaluating NK cells ADCC. Graphic abstract: Workflow of stimulating NK cells via CD16 by plate-bound anti-CD16 mAb.

Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

Three-dimensional (3D) cell culture models are widely used in tumor studies to more accurately reflect cell-cell interactions and tumor growth conditions in vivo. 3D anchorage-independent spheroids derived by culturing cells in ultra-low attachment (ULA) conditions is particularly relevant to ovarian cancer, as such cell clusters are often observed in malignant ascites of late-stage ovarian cancer patients. We and others have found that cells derived from anchorage-independent spheroids vary widely in gene expression profiles, proliferative state, and metabolism compared to cells maintained under attached culture conditions. This includes changes in mitochondrial function, which is most commonly assessed in cultured live cells by measuring oxygen consumption in extracellular flux assays. To measure mitochondrial function in anchorage-independent multicellular aggregates, we have adapted the Agilent Seahorse extracellular flux assay to optimize measurements of oxygen consumption and extracellular acidification of ovarian cancer cell spheroids generated by culture in ULA plates. This protocol includes: (i) Methods for culturing tumor cells as uniform anchorage-independent spheroids; (ii) Optimization for the transfer of spheroids to the Agilent Seahorse cell culture plates; (iii) Adaptations of the mitochondrial and glycolysis stress tests for spheroid extracellular flux analysis; and (iv) Suggestions for optimization of cell numbers, spheroid size, and normalization of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values. Using this method, we have found that ovarian cancer cells cultured as anchorage-independent spheroids display altered mitochondrial function compared to monolayer cultures attached to plastic dishes. This method allows for the assessment of mitochondrial function in a more relevant patho/physiological culture condition and can be adapted to evaluate mitochondrial function of various cell types that are able to aggregate into multicellular clusters in anchorage-independence. Graphic abstract: Workflow of the Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI.

Mechanisms that target and destroy foreign nucleic acids are major barriers to horizontal gene transfer (HGT) in prokaryotes. Amongst them, restriction-modification (R-M) systems are found in ≥75% of the sequenced genomes in Bacteria and Archaea. Due to their high target sequence specificity and potent nucleolytic activity, R-M systems are used as a paradigm to elucidate the mechanisms of DNA binding and cleavage. Since these enzymes modulate HGT, they are one of the machineries implicated in the ability of a bacterium to gain antibiotic resistance. This protocol provides a detailed purification strategy for the Type IV restriction endonuclease SauUSI from Staphylococcus aureus. This protocol eventually leads to ≥95% purity of protein which can then be used for crystallographic and biochemical purposes. Graphic abstract: Workflow for purification of SauUSI.

Tissue-Specific, Genome-wide Mapping of R-loops in Drosophila Using MapR.

R-loops, or RNA:DNA hybrids, are structures that arise co-transcriptionally when a nascent RNA hybridizes back with the template ssDNA, leading to a displaced ssDNA. Because accumulation of R-loops can lead to genomic instability and loss of cellular homeostasis, it is important to determine the genome-wide distribution of R-loops in different physiological conditions. Current R-loop mapping strategies are based on R-loop enrichment-mediated by the S9.6 antibody, such as DRIP-seq, or by the exonuclease RNase H1, such as MapR-or the latest R-loop CUT&Tag, based on an artificial R-loop sensor derived from an RNase H1 sub-domain. Because some of these techniques often require high input material or expensive reagents, we sought to apply MapR, which does not require expensive reagents and has been shown to be compatible with low input samples. Importantly, we demonstrate that incorporation of improved CUT&RUN steps into the MapR protocol yields R-loop-enriched DNA when using low input Drosophila nuclei. Graphical abstract: Workflow for mapping tissue-specific, genome-wide R-loops in Drosophila . Purify GST-tagged and catalytically inactive RNase H1 tethered MapR enzymes, GST-ΔRH-MNase, and GST-MNase, from transformed E. coli. Perform tissue-specific nuclei immuno-enrichment from UAS-EGFP.KASH-Msp300 Drosophila using magnetic bead-bound green fluorescent protein (GFP) antibody. Incubate isolated nuclei with MapR enzymes and activate MNase DNA cleavage with low salt/high calcium buffers. Purify released, R-loopenriched DNA fragments and generate sequencing-ready libraries. Align MapR data to reference genome and compare R-loop enrichment peaks in genome browser.

Three-dimensional Models of the Nasopharynx for the Study of Epstein-Barr Virus Infection.

The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods ( Ziegler et al., 2021 ). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures. Graphic abstract: Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.

Design and implementation of customized workflow configuration platform for electric power company

As power supply company carries out work to optimize the business environment, much problems such as asymmetric information about electricity consumption of electricity users, poor professional interaction channels and low work efficiency have emerged while the level of power supply services has been improved. Relying on the Internet of Things (IoT) platform of power supply company, open graphic library (OpenGL) technology, Activiti engine and Java programming technology and other mature technologies, a set of graphical workflow configuration platform has been built to support the customization of the electricity users. The platform graphically implements the construction of power business workflow, using drag-and-drop components and model-driven logic to create front-end web and mobile applications. The workflow platform conducts compliance audit during workflow configuration, keeps business information strictly outside the negative list, and each work is automatically initiated, executed and associated to abstract behaviours with high application rate and comprehensiveness based on rich and diverse work scenarios to meet the needs of Beibei power supply company. After the workflow configuration platform was applied, the business management and execution efficiency of the power supply company was improved by more than 30%.

A graphical, interactive and GPU-enabled workflow to process long-read sequencing data

BackgroundLong-read sequencing has great promise in enabling portable, rapid molecular-assisted cancer diagnoses. A key challenge in democratizing long-read sequencing technology in the biomedical and clinical community is the lack of graphical bioinformatics software tools which can efficiently process the raw nanopore reads, support graphical output and interactive visualizations for interpretations of results. Another obstacle is that high performance software tools for long-read sequencing data analyses often leverage graphics processing units (GPU), which is challenging and time-consuming to configure, especially on the cloud.ResultsWe present a graphical cloud-enabled workflow for fast, interactive analysis of nanopore sequencing data using GPUs. Users customize parameters, monitor execution and visualize results through an accessible graphical interface. The workflow and its components are completely containerized to ensure reproducibility and facilitate installation of the GPU-enabled software. We also provide an Amazon Machine Image (AMI) with all software and drivers pre-installed for GPU computing on the cloud. Most importantly, we demonstrate the potential of applying our software tools to reduce the turnaround time of cancer diagnostics by generating blood cancer (NB4, K562, ME1, 238 MV4;11) cell line Nanopore data using the Flongle adapter. We observe a 29x speedup and a 93x reduction in costs for the rate-limiting basecalling step in the analysis of blood cancer cell line data.ConclusionsOur interactive and efficient software tools will make analyses of Nanopore data using GPU and cloud computing accessible to biomedical and clinical scientists, thus facilitating the adoption of cost effective, fast, portable and real-time long-read sequencing.

IoT-Driven Workflows for Risk Management and Control of Beehives

The internet of things (IoT) and Industry 4.0 technologies are becoming widely used in the field of apiculture to enhance honey production and reduce colony losses using connected scales combined with additional data, such as relative humidity and internal temperature. This paper exploits beehive weight measurements and builds appropriate business rules using two instruments. The first is an IoT fixed scale installed on one hive, taking rich continuous measurements, and used as a reference. The second is a portable nomad scale communicating with a smartphone and used for the remaining hives. A key contribution will be the run and triggering of a business process model based on apicultural business rules learned from experience and system observed events. Later, the evolution of the weight of each individual hive, obtained by either measurement or inference, will be associated with a graphical workflow diagram expressed with the business process model and notation (BPMN) language, and will trigger events that inform beekeepers to initiate relevant action. Finally, the BPMN processes will be transformed into executable models for model driven decision support. This contribution improves amateur and professional user-experience for honeybee keeping and opens the door for interoperability between the suggested model and other available simulations (weather, humidity, bee colony behavior, etc.).

Isolation of Nasal Brush Cells for Single-cell Preparations.

Solitary chemosensory epithelial cells are scattered in most mucosal surfaces. They are referred to as tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. They are the primary source of IL-25 in the epithelium and are also engaged in acetylcholine generation. We recently demonstrated that nasal solitary chemosensory (brush) cells can generate robust levels of cysteinyl leukotrienes in response to stimulation with calcium ionophore, aeroallergens, and danger-associated molecules, such as ATP and UTP, and this mechanism depends on brush cell expression of the purinergic receptor P2Y2. This protocol describes an effective method of nasal brush cell isolation in the mouse. The method is based on physical separation of the mucosal layer of the nasal cavity and pre-incubation with dispase, followed by digestion with papain solution. The single cell suspension obtained this way contains a high yield of brush cells for fluorescence-activated cell sorting (FACS), RNA-sequencing, and ex vivo assays. Graphic abstract: Workflow of nasal digestion for brush cell isolation.

Preparation of Drosophila Larval Blood Cells for Single-cell RNA Sequencing.

Recent advances in single-cell RNA-sequencing (scRNA-seq) technologies provide unprecedented opportunities to identify new cell types and characterize cell states. One of the most important requirements for performing scRNA-seq is to obtain high-quality single cells in suspension. Recently, we used this approach to characterize Drosophila blood cells (hemocytes). Here, we provide a detailed protocol for obtaining single hemocytes in suspension, which can be used for microfluidics-based scRNA-seq platforms. This protocol involves the simple bleeding of third instar larvae and the subsequent purification of the hemolymph using either Optiprep-based gradient centrifugation or traditional centrifugation methods to obtain single hemocytes of high quality for scRNA-seq. Importantly, this method for single-hemocyte preparation is straightforward and reproducible, with negligible issues associated with cell viability as the entire procedure involves no enzymatic dissociation. Graphic abstract: Workflow for the preparation of Drosophila larval blood cells in suspension. Hemocytes (blood cells) of the sessile and circulatory compartments of larvae are derived by simple bleeding and purification using gradient centrifugation. Blood cells are counted and subsequently encapsulated by microfluidics-based scRNA-seq platforms. Blood cells represented in the schematic are derived from third instar larvae of the genotype Hemolectin-GAL4.Delta, UAS-2xEGFP (BDSC stock #30140).

Delivery of AAV for Expression of Fluorescent Biosensors in Juvenile Mouse Hippocampus.

Genetically encoded fluorescent biosensors are versatile tools for studying brain metabolism and function in live tissue. The genetic information for these biosensors can be delivered into the brain by stereotaxic injection of engineered adeno-associated viruses (AAVs), which can selectively target different cell types depending on the capsid serotype and/or the viral promoter. Here, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD+ ratio and the intracellular Ca2+ levels in individual dentate granule cells from acute hippocampal slices. Graphic abstract: Workflow diagram for biosensor expression in the mouse hippocampus using intracranial injections of adeno-associated viruses.

Extraction and Electrophoretic Analysis of Bacterial Lipopolysaccharides and Outer Membrane Proteins.

Lipopolysaccharides (LPS) (or lipooligosaccharides [LOS], which lack the O-antigen side chains characteristic of LPS), and outer membrane proteins (OMP) are major cell-surface molecules in the outer membrane (OM) of gram-negative bacteria. The LPS is responsible for causing endotoxic shock in infected hosts and, in conjunction with some OMPs, provides protection to the bacterium against host innate immune defenses and attachment to host cells. Electrophoretic analysis can provide valuable information regarding the size, number, and variability of LPS/LOS and OMP components between bacterial strains and mutants, which aids in understanding the basic biology and virulence factors of a particular species. Furthermore, highly purified extracts are normally not required if only electrophoretic analysis is to be done, and various methods have been established for such procedures. Here, we review ameliorated procedures for fast and convenient extraction of LPS/LOS and protein-enriched outer membranes (PEOM) for optimal electrophoretic resolution. Specifically, we will describe the phenol-based micro-method for LPS/LOS extraction, a differential extraction procedure with sodium lauryl sarcosinate for PEOM, and gel preparation for electrophoretic analysis of LPS/LOS samples in detail. Graphic abstract: Workflow for the preparation and analysis of LPS/LOS and PEOM.

Optimized Immunostaining of Embryonic and Early Postnatal Mouse Brain Sections.

The mammalian neocortex, the outer layer of the cerebrum and most recently evolved brain region, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the protein expression of graded transcription factors and molecular determinants that define the identity of different projection neurons. Thus, specific detection and visualization of protein expression is crucial for assessing the identity of neocortical neurons and, more broadly, for understanding early and late developmental mechanisms and function of this complex system. Several immunostaining/immunofluorescence methods exist to detect protein expression. Published protocols vary with regard to subtle details, which may impact the final outcome of the immunofluorescence. Here, we provide a detailed protocol, suitable for both thin cryostat sections and thick vibratome sections, which has successfully worked for a wide range of antibodies directed against key molecular players of neocortical development. Ranging from early technical steps of brains collection down to image analysis and statistics, we include every detail concerning sample inclusion and sectioning, slide storage and optimal antibody dilutions aimed at reducing non-specific background. Routinely used in the lab, our background-optimized immunostaining protocol allows efficient detection of area- and layer- specific molecular determinants of distinct neocortical projection neurons. Graphic abstract: Workflow chart for the optimized immunostaining protocol of mouse brain sections. A. A flow chart for different steps of the optimized immunostaining protocol on both thin cryostat and thick vibratome sections. B. Example for immunostaining against Satb2 and Ctip2 on a thin coronal section (20 μm) at the level of the somatosensory cortex. The first column to the left shows the binning system where 6 bins can be overlaid on the image. On the bottom, an example of counting analysis showing the percentage of marker-positive cells normalized to the total number of DAPI or Hoechst-positive cells. C. Example for immunostaining against Satb2 and Ctip2 on a GFP+ thick vibratome section (200 μm). Images are taken at low magnification (10x, left) and high magnification (40x, right). The graph shows a counting of the percentage of Ctip2-positive neurons normalized to the total number of GFP-electroporated neurons on high-magnification images. Images on B and C are modified from Harb et al. (2016).

Generation and Implementation of Reporter BHK-21 Cells for Live Imaging of Flavivirus Infection.

The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus genetically-encoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe the image analysis procedures and provide the required software pipelines. Our reporter cells allow the implementation of single-cell infection kinetics as well as plaque assays for both reference and native strains of flaviviruses by live-cell imaging. Graphic abstract: Workflow for the generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection.