Multiwell Plates Research Articles

Method to determine the decolorization potential of persistent dyes by white rot fungi by colorimetric assays

Decolorization assays allow to assess the ability of white rot fungi to degrade persistent organic molecules such as textile dyes and can contribute to discover microorganisms that can be used for bioremediation. The decolorization can be overlayed by the absorption from metabolites that are produced by fungi during screening, which interfere with the results. To compensate for this interference a method was developed by using different controls to subtract interfering signals.The method was designed for simple screening in multiwell plates that can be operated with a plate reader. It was applied to four different textile dyes (Reactive Black 5, Reactive Blue 4, Reactive Green 19, and Reactive Orange 16) that were degraded by the white rot fungus Phanerochaete velutina. The four textile dyes showed different results with a different degree of interference. The controls allow to compensate for interfering signals and to calculate kinetic parameters for the decolorization reaction and the enzymatic degradation.•Determine the non-enzymatic degradation of the dyes in experiments without fungi.•Determine the absorbance of metabolites and subtract it from the decolorization data to obtain the degradation of the dye.•Determine kinetic parameters of the degradation to compare the efficiency of the enzymes towards dyes.

Deep Learning Image Analysis of High-Throughput Toxicology Assay Images.

High-throughput chemical screening approaches often employ microscopy to capture photomicrographs from multi-well cell culture plates, generating thousands of images that require time-consuming human analysis. To automate this subjective and time-consuming manual process, we have developed a method that uses deep learning to automatically classify digital assay images. We have trained a convolutional neural network (CNN) to perform binary and multi-class classification. The binary classifier binned assay images into healthy (comparable to untreated controls) and altered (not comparable to untreated-control) classes with >98% accuracy; the multi-class classifier assigned "Healthy," "Intermediate" and "Altered" labels to assay images with >95% accuracy. Our dataset comprised high-resolution assay images from primary human hepatocytes and undifferentiated (proliferating) and differentiated 2D cultures of HepaRG cells. In this study we have focused on testing and fine-tuning various CNN architectures, including ResNet 34, 50 and 101. To visualize regions in the images that the CNN model used for classification, we employed Class Activation Maps (CAM). This allowed us to better understand the inner workings of the neural network and led to additional optimizations of the algorithm. The results indicate a strong correspondence between dosage and classifier-predicted scores, suggesting that these scores might be useful in further characterizing benchmark dose. Together, these results clearly demonstrate that deep-learning based automated image classification of cell morphology changes upon chemical-induced stress can yield highly accurate and reproducible assessments of cytotoxicity across a variety of cell types.

Aqueous broadband photopolymerization on microreactor arrays: from high throughput polymerization to fabricating artificial cells

Microreactor arrays combining ZnO and polyaniline are fabricated onto the bottom of multi-well plates to catalyze broadband sunlight-driven open-to-air polymerization in aqueous media.

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3′-phosphate-5′-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3′,5′-diphosphate) as a side product. We present a europium(iii)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

Luciferase-Based Determination of ATP/NAD(H) Pools in a Marine (Environmental) Bacterium

In all living organisms, adenosine triphosphate (ATP) and NAD(H) represent universal molecular currencies for energy and redox state, respectively, and are thus widely applicable molecular proxies for an organism’s viability and activity. To this end, corresponding luciferase-based assays in combination with a microplate reader were established with the marine model bacterium Phaeobacter inhibens DSM 17395 (Escherichia coli K12 served as reference). Grey multiwell plates best balanced sensitivity and crosstalk, and optimal incubation times were 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD⁺, and NADH, respectively. Quenching of bacterial cell samples involved Tris-EDTA-DTAB and bicarbonate base-DTAB for ATP and NAD(H) assays, respectively. The ATP and NAD(H) yields determined for P. inhibens DSM 17395 at ¼ OD_max were found to reside well within the range previously reported for E. coli and other bacteria, e.g., 3.28 µmol ATP (g cells_dry)⁻¹. Thus, the here described methods for luciferase-based determination of ATP/NAD(H) pools open a promising approach to investigate energy and redox states in marine (environmental) bacteria.

Encapsulated droplet interface bilayers as a platform for high-throughput membrane studies.

Whilst it is highly desirable to produce artificial lipid bilayer arrays allowing for systematic high-content screening of membrane conditions, it remains a challenge due to the combined requirements of scaled membrane production, simple measurement access, and independent control over individual bilayer experimental conditions. Here, droplet bilayers encapsulated within a hydrogel shell are output individually into multi-well plates for simple, arrayed quantitative measurements. The afforded experimental throughput is used to conduct a 2D concentration screen characterising the synergistic pore-forming peptides Magainin2 and PGLa. Maximal enhanced activity is revealed at equimolar peptide concentrations via a membrane dye leakage assay, a finding consistent with models proposed from NMR data. The versatility of the platform is demonstrated by performing in situ electrophysiology, revealing low conductance pore activity (∼15 to 20 pA with 4.5 pA sub-states). In conclusion, this array platform addresses the aforementioned challenges and provides new and flexible opportunities for high-throughput membrane studies. Furthermore, the ability to engineer droplet networks within each construct paves the way for “lab-in-a-capsule” approaches accommodating multiple assays per construct and allowing for communicative reaction pathways.

The use of zebrafish to evaluate neuropharmacology of the gold nanoparticles

Zebrafish (Danio rerio) is a vertebrate animal used in animal model research with complex brains and behaviors similar to humans and associate with low coast become a model attractive for the academic community to seek zebrafish for scientific research. Studies on diseases of the central nervous system (CNS) have advanced and news therapeutic agents were developed for treatment these disorders. Reports suggest that the zebrafish model supports the neurodegenerative studies due functional conservation between human genes implicated in neurodegenerative disorders. The discovery of therapeutic compounds for CNS using the zebrafish model allows to show a neuroprotective action or neurotoxicity that might alter the behavioral changes. Neurotoxicity tests might perform in zebrafish’s embryos into 96 multi-well plates, which reduces the amount of substances used and cost. The bioactive compounds able to penetrate the blood-brain barrier (BBB) have important role physicochemical properties that might be desirable pharmacological effects and zebrafish trials allow if the substances might penetrate BBB and to exert central activity. The assays zebrafish are used to analyze nanoparticles that are small molecules used to explore variety applications in human health. Gold nanoparticles (AuNPs) has important properties which are extremely interest for pharmaceutical area such as drug delivery, cellular imaging, diagnostics, and therapeutic agents. Gold nanoparticles enhances Parkinson symptoms and improved neuroinflammation. Some studies show zebrafish might use to evaluate gold nanoparticles for human health hazard and toxicity studies. There is enormous potential for zebrafish in preclinical assays due to predict pharmacological and toxicity effects. Specific guidelines focused on methodologies in the zebrafish are needed to ensure adequate reproducible trials.

A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium

Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid cycle). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research.

First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA.

Simple SummaryRNA interference (RNAi), a conserved mechanism in eukaryotic organisms, is initiated by the presence of double-stranded RNA (dsRNA) in the cells, regulating the expression of specific genes by degradation of their mRNA, and it is known as post-transcriptional gene-silencing (PTGS). RNAi can be employed to develop species-specific biopesticides where dsRNA is delivered to a pest insect via the oral route. Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound. This study reports an artificial diet-based multi-well plate method and the first findings on the potential application of biotechnology in the control of the banana weevil using dietary RNAi, which would arguably provide the most sustainable and practical method for dsRNA delivery. A selection of target genes with effective RNAi leading to high and rapid mortality is presented. These results indicate the potential of RNAi-mediated management to suppress C. sordidus efficiently in the banana crop.Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9–16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3–4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

Using Microfluidics to Generate Human Naïve and Primed Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) are generated from somatic cells by the expression of a cocktail of transcription factors, and iPSCs have the capacity to generate in vitro all cell types of the human body. In addition to primed (conventional) iPSCs, several groups recently reported the generation of human naïve iPSCs, which are in a more primitive developmental state and have a broader developmental potential, as shown by their ability to form cells of the placenta. Human iPSCs have broad medical potential but their generation is often time-consuming, not scalable and requires viral vectors or stable genetic manipulations. To overcome such limitations, we developed protocols for high-efficiency generation of either conventional or naïve iPSCs by delivery of messenger RNAs (mRNAs) using a microfluidic system. In this protocol we describe how to produce microfluidic devices, and how to reprogram human somatic cells into naïve and primed iPSCs using these devices. We also describe how to transfer the iPSC colonies from the microfluidic devices over to standard multiwell plates for subsequent expansion of the cultures. Our approach does not require stable genetic modifications, is reproducible and cost-effective, allowing to produce patient-specific iPSCs for cell therapy, disease modeling, and in vitro developmental studies.

Methods for rapid prototyping novel labware: using CAD and desktop 3D printing in the microbiology laboratory.

Although the microbiology laboratory paradigm has increasingly changed from manual to automated procedures, and from functional to molecular methods, traditional culture methods remain vital. Using inexpensive desktop fused filament fabrication 3D printing, we designed, produced and tested rapid prototypes of customised labware for microbial culture namely frames to make dip slides, inoculation loops, multi-pin replicators, and multi-well culture plates for solid medium. These customised components were used to plate out samples onto solid media in various formats, and we illustrate how they can be suitable for many microbiological methods such as minimum inhibitory concentration tests, or for directly detecting pathogens from mastitis samples, illustrating the flexibility of rapid-prototyped culture consumable parts for streamlining microbiological methods. We describe the methodology needed for microbiologists to develop their own novel and unique tools, or to fabricate and customise existing consumables. A workflow is presented for designing and 3D printing labware and quickly producing easy-to-sterilise and re-useable plastic parts of great utility in the microbiology laboratory.

Abstract 14161: Drug Compounds Combination Screening Using Human Induced Pluripotent Stem Cell-Derived Cardiac Tissues

Introduction: The introduction of human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) technology provided an important tool for the field of safety pharmacology (QT screening). This approach has been limited, however, mostly to the assessment of a single drug. We present a large scale EP phenotyping system comprised of hiPSC-derived cardiac cell sheets (hiPSC-CCSs), fluorescent voltage sensors and actuators, and an optical mapping system; with the aim of performing drug combination screening. Methods and Results: hIPSCs-CMs were seeded as hiPSC-CCSs in multi-well plates. Tissues were treated with varying concentrations of drug compounds’ combinations. Optical signals were acquired following loading of the hiPSC-CCSs with voltage-sensitive dyes or expression of genetically-encoded voltage indicator (ArcLight). EP parameters such as conduction velocity (CV) and action potential duration (APD) were extracted. ATX2 (late sodium current activator) and Dofetilide (IKr blocker) were chosen as representative APD prolonging reagents. Example of the effects of 16 different drug doses combinations on APD (% change) prolongation is depicted in Fig.1a. Similarly, examples of the restitution plots obtained at 4 different drug combinations are presented in Fig.1b-c. Notice that drug compound's effect on prolonging APD showed greater effect as compared to the cumulative effect of each drug alone. Figure 1: (a) Percentage of change in APD 80 15 minutes post treatment (b, c) APD 80 per condition before and after treatment, respectively Conclusion: These results highlight the potential of the hiPSC-CCS as a model for combinational drug screening. This may bring a unique value to the fields of safety pharmacology, pathophysiology testing and drug screening applications.

Optical deciphering of multinary chiral compound mixtures through organic reaction based chemometric chirality sensing

The advances of high-throughput experimentation technology and chemometrics have revolutionized the pace of scientific progress and enabled previously inconceivable discoveries, in particular when used in tandem. Here we show that the combination of chirality sensing based on small-molecule optical probes that bind to amines and amino alcohols via dynamic covalent or click chemistries and powerful chemometric tools that achieve orthogonal data fusion and spectral deconvolution yields a streamlined multi-modal sensing protocol that allows analysis of the absolute configuration, enantiomeric composition and concentration of structurally analogous—and therefore particularly challenging—chiral target compounds without laborious and time-consuming physical separation. The practicality, high accuracy, and speed of this approach are demonstrated with complicated quaternary and octonary mixtures of varying chemical and chiral compositions. The advantages over chiral chromatography and other classical methods include operational simplicity, increased speed, reduced waste production, low cost, and compatibility with multiwell plate technology if high-throughput analysis of hundreds of samples is desired.

Prediction of Clinical Response to Venetoclax Plus Decitabine in AML Using a 7-Day Ex Vivo Assay

Prediction of Clinical Response to Venetoclax Plus Decitabine in AML Using a 7-Day Ex Vivo Assay

Rapid 3D BioPrinting of a human iPSC-derived cardiac micro-tissue for high-throughput drug testing

With cardiac disease a reigning problem in the world, the need for accurate and high-throughput drug testing is paramount. 3D cardiac tissues are promising models, as they can recapitulate the cell-cell, cell-matrix, and cell-tissue interactions that impact response to a drug. Using an in-house developed micro-continuous optical printing system, we created a cardiac micro-tissue in mere seconds with microscale alignment cues in a hydrogel scaffold that is small enough to fit in a 96-well plate. The 3D printed, asymmetric, cantilever-based tissue scaffold allows one to directly measure the deformation produced by the beating micro-tissue. After 7 days, the micro-tissue exhibited a high level of sarcomere organization and a significant increase in maturity marker expression. The cardiac micro-tissues were validated against two representative drugs, isoproterenol and verapamil at various doses, showing corresponding and measurable changes in beating frequency and displacement. Such rapidly bioprinted cardiac micro-tissues in a multi-well plate offer a promising solution for high-throughput screening in drug discovery.

Multiplexed single-cell proteomics using SCoPE2.

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

A machine learning-enhanced biosensor for mercury detection based on an hydrophobin chimera

Marine waters are becoming contaminated by diverse pollutants at a fast rate, and detection of these water pollutants has become a major concern in recent years. Among these, mercury is considered the most toxic element for human health. At present, despite the commonly used methods for its detection are accurate, they often require sophisticated equipments, have relatively high costs, are demanding and time-consuming.Herein a novel solution to detect mercury (II) pollution in sea water is proposed, and an easy and portable detection method has been developed. Indeed, a hydrophobin based chimera able to both adhere to polystyrene multiwell plates and bind mercury (II) with a consequent fluorescent decrease was designed. The chimera was the recognition element in a fluorescence-based biosensor able to detect mercury (II) in the nM range. Indeed, this biosensor specifically measure Hg2+ concentration also in the presence of other metals, reaching a limit of detection of 0.4 nM in tap water and 0.3 nM in sea water. Moreover, the developed biosensor was coupled to machine learning methodologies with the big advantage of predicting mercury concentration levels without the use of classical reader devices, thus allowing in situ monitoring of sea pollution by non-skilled personnel.

Rapid Analysis of Volatile Phenols from Grape Juice by Immersive Sorbent Sheet Extraction Prior to Direct Analysis in Real-Time Mass Spectrometry (DART-MS).

Poly(dimethylsiloxane)-based thin-film sorbent sheets (SPMESH) have previously been used for parallel headspace (HS) extraction prior to direct analysis in real-time mass spectrometry (DART-MS) for rapid quantitation of odorants in complex matrices. However, HS-SPMESH extraction is poorly suited for less volatile odorants, e.g., volatile phenols. This report describes modifications to the previous SPMESH extraction device, which make it amenable to parallel extraction of low-volatility analytes from multiwell plates under direct immersion (DI) conditions. Optimization and validation of the DI-SPMESH-DART-MS approach were performed on four volatile phenols (4-ethylphenol, 4-ethylguaiacol, 4-methylguaiacol, and guaiacol) of relevance to the quality of grape juices. Negative-ion mode DART-MS spectra showed a series of oxygenated adducts [M + nO - H]- for all analytes, but isobaric interferences could be limited for three of the four analytes by selecting an appropriate MS/MS transition. Signal suppression from nonvolatiles (sugars, acids) could be overcome by a rinse step. DI-SPMESH-DART-MS analysis of 24 samples could be performed in ∼45 min (30 min extraction, 16 min DART analysis) with 0.5-3 μg/L detection limits in aqueous and model juice solutions. In real grape juices (n = 5 cultivars), good accuracy (72-137%) could be achieved for two of the four volatile phenols initially investigated, 4-ethylphenol and 4-ethylguaiacol. However, poor accuracy was observed for guaiacol in some cultivars, and 4-methylguaiacol could not be quantitated due to interferences with other volatile phenols. Despite these limitations, DI-SPMESH-DART-MS/MS may be useful for prescreening a large number of samples prior to more selective conventional analyses.

A High-Throughput Assay for Quantifying Phenotypic Traits of Microalgae.

High-throughput methods for phenotyping microalgae are in demand across a variety of research and commercial purposes. Many microalgae can be readily cultivated in multi-well plates for experimental studies which can reduce overall costs, while measuring traits from low volume samples can reduce handling. Here we develop a high-throughput quantitative phenotypic assay (QPA) that can be used to phenotype microalgae grown in multi-well plates. The QPA integrates 10 low-volume, relatively high-throughput trait measurements (growth rate, cell size, granularity, chlorophyll a, neutral lipid content, silicification, reactive oxygen species accumulation, and photophysiology parameters: ETRmax, Ik, and alpha) into one workflow. We demonstrate the utility of the QPA on Thalassiosira spp., a cosmopolitan marine diatom, phenotyping six strains in a standard nutrient rich environment (f/2 media) using the full 10-trait assay. The multivariate phenotypes of strains can be simplified into two dimensions using principal component analysis, generating a trait-scape. We determine that traits show a consistent pattern when grown in small volume compared to more typical large volumes. The QPA can thus be used for quantifying traits across different growth environments without requiring exhaustive large-scale culturing experiments, which facilitates experiments on trait plasticity. We confirm that this assay can be used to phenotype newly isolated diatom strains within 4 weeks of isolation. The QPA described here is highly amenable to customisation for other traits or unicellular taxa and provides a framework for designing high-throughput experiments. This method will have applications in experimental evolution, modelling, and for commercial applications where screening of phytoplankton traits is of high importance.

Evaluating the in vitro Cytotoxicity of Thymus vulgaris Essential Oil on MCF-7 and HeLa Cancer Cell Lines

Thyme essential oil (TEO) was extracted from dried leaves of Thymus vulgaris. The air-dried aerial parts of the plant produced 1.0% yield of TEO. The detection of this essential oil’s compounds was performed by GC-MASS. The cytotoxic activity of TEO was evaluated against two human cancer cell lines, namely HeLa (human epithelial cervical cancer) and MCF-7 (human breast carcinoma). Cells grown in 96 multi-well plates were treated with six concentrations of EO (6.25, 12.5, 25, 50, 100, 200 ppm) and incubated at 37 °C for 72 hrs. Cancer cell lines elicited various degrees of sensitivity to the cytotoxic effect of essential oil. The TEO exhibited significant differences (p≤ 0.01) between the effects of all concentrations against these two human cell lines. The results showed the highest toxicity of TEO on HeLa cell line (78.67%) and MCF-7 cell line (83.60%) at 200 ppm concentration. Also the values of half-maximal inhibitory concentration (IC50) of TEO against HeLa and MCF-7 cell lines were 34.63 and 27.66 ppm, respectively. Cells treated with the IC50 of TEO showed a significant difference (p ≤ 0.01) in p53 fold expression between HeLa cell line (4.33±0.41 folds) and MCF-7 cell line (5.10±0.32 folds). In general, a dose-dependent decrease the survival of the two cell lines was observed. In addition, MCF-7 cell line revealed higher sensitivity against TEO than HeLa cell line.