Multiwell Plates Research Articles

250 A Living Library for Uveal Melanoma

OBJECTIVES/GOALS: Overall response rates for metastatic uveal melanoma (UM), regardless of treatment modality, are less than 20%, highlighting an urgent need for novel therapies. Herein, we present a UM patient-derived organoid (PDO) biobank as a novel platform for translational research. METHODS/STUDY POPULATION: Patients with primary choroidal or ciliochoroidal UM undergoing enucleation from 7/1/2019-9/30/2022 were invited to enroll. Tumor tissue was harvested within 30 minutes of globe removal. Cells were isolated using the human tumor isolation kit and gentleMACS dissociation protocol (Miltenyi Biotech). PDOs were placed on Cultrex-coated multiwell plates and cultured in supplemented RPMI media. DNA and RNA were isolated using kits from Zymo Research. Exon-enriched libraries and RNA were sequenced using an Illumina HiSeq 4000. Immunohistochemistry (IHC) assessed the following histone post-translational modifications: H3K4me1/3, H3K27Ac, and H3K27me. RESULTS/ANTICIPATED RESULTS: PDOs were established in 19 of 20 (95%) attempted cases. BAP1 protein expression was retained (n=7) or lost (n=12) in the primary tumors, with matching phenotype confirmed in PDOs. In 9 sequenced cases, a driving mutation was present in GNAQ (n=4), GNA11 (n=4), or CYSLTR2 (n=1). Morphology ranged from spindle-like to epithelioid clusters, mimicking primary tumor histopathology. Pigmentation increased with time in culture. Growth in culture was slow, and 1-2 months were allotted prior to passaging in most cases. Whole exome and RNA-sequencing confirmed distinct molecular profiles, with differential staining of active chromatin marks by IHC. DISCUSSION/SIGNIFICANCE: A biobank of primary UM PDOs with unique morphological and molecular characteristics has been established. These will serve as a model of human disease to facilitate translational research and investigate personalized treatments for patients with UM.

Commercial dishes with gelatin-free microstructured inserts for elongated stem cell self-renewal and pluripotency

Here, we report the scalable fabrication of 2i-functionalized micro-pyramid-array (μPyA/+2i) inserts for use in commercial multi-well plates, as the alternative cultivation platform for maintaining long-term self-renewal and pluripotency of multiple mESCs and mouse induced pluripotent stem cells. Relevant evidence including cell morphology characterization increased alkaline phosphatase activity, high expression of mESC self-renewal markers, decreased levels of differentiation-associated markers, and high proportion of self-renewal marker cells are provided. Further studies demonstrated that μPyA/+2i could cause a higher cell density in mESC colony, and induce gene expression changes. Subsequent studies showed that μPyA/+2i can influence the cytoskeleton and promote cell adhesion through Cldn-7 upregulation. In summary, these μPyA/+2i inserts offer flexible and gelatin-free micro-envriomnets to maintain long-term self-renewal and pluripotency of mESCs. Enabled by the microstructured inserst, the facile stem cell manipulation and transfer among culture dishes will broaden stem cells both in routine and translational applications.

Colorimetric ortho-aminobenzaldehyde assay developed for the high-throughput chemical screening of inhibitors against dihydrodipicolinate synthase from pathogenic bacteria

In search of a new class of antibacterial agents, compounds that target the essential bacterial enzyme, dihydrodipicolinate synthase (DHDPS), are of interest to drug discovery efforts. DHDPS catalyzes the first committed step in the diaminopimelate (DAP) pathway to the biosynthesis of lysine in bacteria and plants. The ortho-aminobenzaldehyde (o-ABA) assay is typically used as a qualitative tool for identifying fractions containing DHDPS during purification. This report is about the development of a high-throughput o-ABA assay format for the quantification of DHDPS enzyme activity using multi-well plates. The colorimetric assay is suitable for determining enzymatic parameters (KM and Vmax) and identifying inhibitors of DHDPS in a high-throughput screen.

Amplification-Free, High-Throughput Nanoplasmonic Quantification of Circulating MicroRNAs in Unprocessed Plasma Microsamples for Earlier Pancreatic Cancer Detection.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that is often detected at an advanced stage. Earlier diagnosis of PDAC is key to reducing mortality. Circulating biomarkers such as microRNAs are gaining interest, but existing technologies require large sample volumes, amplification steps, extensive biofluid processing, lack sensitivity, and are low-throughput. Here, we present an advanced nanoplasmonic sensor for the highly sensitive, amplification-free detection and quantification of microRNAs (microRNA-10b, microRNA-let7a) from unprocessed plasma microsamples. The sensor construct utilizes uniquely designed -ssDNA receptors attached to gold triangular nanoprisms, which display unique localized surface plasmon resonance (LSPR) properties, in a multiwell plate format. The formation of -ssDNA/microRNA duplex controls the nanostructure-biomolecule interfacial electronic interactions to promote the charge transfer/exciton delocalization processes and enhance the LSPR responses to achieve attomolar (10-18 M) limit of detection (LOD) in human plasma. This improve LOD allows the fabrication of a high-throughput assay in a 384-well plate format. The performance of nanoplasmonic sensors for microRNA detection was further assessed by comparing with the qRT-PCR assay of 15 PDAC patient plasma samples that shows a positive correlation between these two assays with the Pearson correlation coefficient value >0.86. Evaluation of >170 clinical samples reveals that oncogenic microRNA-10b and tumor suppressor microRNA-let7a levels can individually differentiate PDAC from chronic pancreatitis and normal controls with >94% sensitivity and >94% specificity at a 95% confidence interval (CI). Furthermore, combining both oncogenic and tumor suppressor microRNA levels significantly improves differentiation of PDAC stages I and II versus III and IV with >91% and 87% sensitivity and specificity, respectively, in comparison to the sensitivity and specificity values for individual microRNAs. Moreover, we show that the level of microRNAs varies substantially in pre- and post-surgery PDAC patients (n = 75). Taken together, this ultrasensitive nanoplasmonic sensor with excellent sensitivity and specificity is capable of assaying multiple biomarkers simultaneously and may facilitate early detection of PDAC to improve patient care.

Higher throughput workflow with sensitive, reliable and automatic quantification of myelination in vitro suitable for drug screening

Multiple sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system (CNS). Immune-mediated myelin and axonal damage that is accompanied by chronic axonal loss causing destruction of the myelin sheaths are hallmarks of MS. While great strides have been made in understanding the molecular underpinnings of re-/myelination, currently no remyelination therapy is available for MS. As myelination is a complex process that is not fully understood, we sought to develop a systematic, reliable, automated and quantitative higher throughput screening method. We aimed to quantitate myelin sheaths in vitro with high sensitivity at the single cell level suitable for testing small compound libraries. To this end, we miniaturised in vitro retinal ganglion cell-oligodendrocyte precursor cell (RGC–OPC) co-cultures into a multi-well plate format. This allowed us to maintain the reciprocal interaction of live axons and oligodendrocytes (OLs) to ensure compact myelin formation. To quantify our co-cultures, we developed a novel computer vision algorithm to precisely measure myelination. We demonstrated efficacy of our system with known pro-differentiating compounds BQ3020 and XAV939 which exhibited robust, efficient, and dose dependent effects on myelination. Through this combination of experimental and technical advances, we have developed a method allowing systematic and reliable testing of remyelinating compound efficacy.

A novel, accurate, and non-invasive liquid biopsy test to measure cellular immune responses as a tool to diagnose early-stage lung cancer: a clinical trials study

IntroductionLung cancer remains the leading cause of death from cancer, worldwide. Developing early detection diagnostic methods, especially non-invasive methods, is a critical component to raising the overall survival rate and prognosis for lung cancer. The purpose of this study is to evaluate two protocols of a novel in vitro cellular immune response test to detect lung cancer. The test specifically quantifies the glycolysis metabolism pathway, which is a biomarker for the activation level of immune cells. It summarizes the results of two clinical trials, where each deploys a different protocol's version of this test for the detection of lung cancer. In the later clinical trial, an improved test protocol is applied.MethodThe test platform is based on changes in the metabolic pathways of the immune cells following their activation by antigenic stimuli associated with Lung cancer. Peripheral Blood Mononuclear Cells are loaded on a multiwell plate together with various lung tumor associated antigens and a fluorescent probe that exhibits a pH-dependent absorption shift. The acidification process in the extracellular fluid is monitored by a commercial fluorescence plate reader device in continuous reading for 3 h at 37 °C to document the fluorescent signal received from each well.ResultsIn the later clinical trial, an improved test protocol was applied and resulted in increased test accuracy. Specificity of the test increased to 94.0% and test sensitivity increased to 97.3% in lung cancer stage I, by using the improved protocol.ConclusionThe improved protocol of the novel cellular immune metabolic response based test detects stage I and stage II of lung cancer with high specificity and sensitivity, with low material costs and fast results.

Polyamide-coated paper-based sorptive phase applied in high-throughput thin film microextraction designed by 3D printing

A new method was developed for the determination of endocrine disrupting compounds (phthalates, alkylphenols and bisphenols) in water samples by using thin film microextraction technique (TFME) with high-performance liquid chromatography and diode-array detection (HPLC-DAD). This paper reports for the first time the use of a paper-based sorptive phase (PSP) in a high-throughput semi-automated system. This system designed by 3D printing technology allowed us to integrate the PSP with a multi-well plate format. Multivariate design of experiments and one-factor-at-a-time approaches were employed for optimization. The optimized PSP preparation required two dips of the filter paper into the nylon 66 solution followed by the evaporation of the solvent. The best conditions for the extraction step were NaCl 30 % (w/v) added to the sample at pH 2 for 180 min. The desorption showed best results with acetonitrile for 20 min. The calibration curves exhibited coefficients of determination higher than 0.9837. The limits of detection varied from 1.5 to 7.6 μg L-1. Six of ten analytes showed promising results with accuracy ranging from 82 to 117 % and precision (RSD) equal or inferior to 24 % for mineral water sample. We also evaluated a coconut water sample that obtained higher deviations than mineral water sample. The time consumed for each sample was about 8.3 min.

Three-Dimensional-Printed Electrochemical Multiwell Plates for Monitoring Food Intolerance from Intestinal Organoids.

Common symptoms of food intolerance are caused by chemical components within food that have a pharmacological activity to alter the motility of the gastrointestinal tract. Food intolerance is difficult to diagnose as it requires a long-term process of eliminating foods that are responsible for gastrointestinal symptoms. Enterochromaffin (EC) cells are key intestinal epithelium cells that respond to luminal chemical stimulants by releasing 5-HT. Changes in 5-HT levels have been shown to directly alter the motility of the intestinal tract. Therefore, a rapid approach for monitoring the impact of chemicals in food components on 5-HT levels can provide a personalized insight into food intolerance and help stratify diets. Within this study, we developed a three-dimensional (3D)-printed electrochemical multiwell plate to determine changes in 5-HT levels from intestinal organoids that were exposed to varying chemical components found in food. The carbon black/poly-lactic acid (CB/PLA) electrodes had a linear range in physiological concentrations of 5-HT (0.1-2 μM) with a limit of detection of 0.07 μM. The electrodes were stable for monitoring 5-HT overflow from intestinal organoids. Using the electrochemical multiwell plate containing intestinal organoids, increases in 5-HT were observed in the presence of 0.1 mM cinnamaldehyde and 10 mM quercetin but reduction in 5-HT levels was observed in 1 mM sorbitol when compared to control. These changes in the presence of chemicals commonly found in food were verified with ex vivo ileum tissue measurements using chromatography and amperometry with boron-doped diamond electrodes. Overall, our 3D electrochemical multiwell plate measurements with intestinal organoids highlight an approach that can be a high-throughput platform technology for rapid screening of food intolerance to provide personalized nutritional diet.

Single-objective selective plane illumination microscopy for multi-well organoid imaging.

Current preclinical in vitro and non-human animal models fail to adequately reproduce human physiology and disease processes. While recent efforts have focused on using microfluidics to help create stem cell-derived organoids as a way to better model 3D vascularization and improve the delivery of therapeutics, there remain challenges especially in terms of long-term imaging. To address this, we report here on the development of single-objective Selective Plane Illumination Microscopy (SoSPIM) for high-content imaging. This platform is capable of imaging samples in different mounting formats, ranging from tissue slices, to conventional culture dishes and and multi-well plates. To demonstrate this platform's capabilities, we imaged cerebral organoid slices and fixed organoids cultured in multi-well microfluidic devices. Our future work will focus on applying SoSPIM for long-term live organoid imaging using machine learning approaches for tracking and mapping intercellular dynamics and structures.

Comparative Bioactivity of Emamectin Benzoate Formulations against the Pine Wood Nematode, Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus is a well-known devastating pathogen of economic importance in the Republic of Korea and other countries. In the Republic of Korea, trunk injection of nematicides is the preferred method of control. In this study, the efficacy of 16 locally produced formulations of emamectin benzoate against the PWN are compared through determining their sublethal toxicities and reproduction inhibition potentials. Nematodes were treated with varying concentrations of the tested chemicals in multi-well culture plates, and rates of paralysis and mortality were determined after 24 h. Reproduction inhibition potential was tested by inoculating pre-treated nematodes onto Botrytis cinerea, and in pine twig cuttings. Despite the uniformity in the concentration of the active ingredient, efficacy was contrastingly different among formulations. The formulations evidently conformed to three distinct groups based on similarities in sublethal activity (group 1: LC95 of 0.00768-0.01443 mg/ml; group 2: LC95 of 0.03202-0.07236 mg/ml, and group 3: LC95 of as high as 0.30643-0.40811 mg/ml). Nematode paralysis generally occurred at the application dose of 0.0134-0.1075 μg/ml, and there were significant differences in nematode paralysis rates among the products. Nematode reproduction was only evident at lower doses both on B. cinerea and pine twigs, albeit the variations among formulations. Group 1 formulations significantly reduced nematode reproduction even at a lower dose of 0.001075 µg/ml. The variations in efficacy might be attributed to differences in inert ingredients. Therefore, there is need to analyze the potential antagonistic effects of the large number of additives used in formulations.

A highly active mineral-based ice nucleating agent supports in situ cell cryopreservation in a high throughput format.

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Methods for Fungicide Efficacy Screenings: Multiwell Testing Procedures for the Oomycetes Phytophthora infestans and Pythium ultimum.

Oomycetes-borne diseases represent a serious problem for agriculture sustainability due to the high use of chemical products employed for their control. In recent years, increasing concerns on side effects associated with fungicide utilization have led to the reduction of the permissible modes of action, with the remaining ones continuously threatened by the increase of resistant strains in the pathogen populations. In this context, it is mandatory to develop new generation fungicides characterized by high specificity towards the target species and low environmental impact to guarantee the sustainability, productivity, and quality of food production. Fungicide discovery is a lengthy and costly process, and despite these urgent needs, poor description and formalization of high-throughput methodologies for screening the efficacy of active compounds are commonly reported for these kinds of organisms. In this study, a comprehensive picture of two high-throughput practices for efficient fungicide screening against plant-pathogenic oomycetes has been provided. Different protocols using multiwell plates were validated on approved crop protection products using Phytophthora infestans and Pythium ultimum as the model species. In addition, detailed statistical inputs useful for the analysis of data related to the efficacy of screenings are included.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

Modification of the swirling well cell culture model to alter shear stress metrics.

Effects of hemodynamic shear stress on endothelial cells have been extensively investigated using the "swirling well" method, in which cells are cultured in dishes or multiwell plates placed on an orbital shaker. A wave rotates around the well, producing complex patterns of shear. The method allows chronic exposure to flow with high throughput at low cost but has two disadvantages: a number of shear stress characteristics change in a broadly similar way from the center to the edge of the well, and cells at one location in the well may release mediators into the medium that affect the behavior of cells at other locations, exposed to different shears. These properties make it challenging to correlate cell properties with shear. The present study investigated simple alterations to ameliorate these issues. Flows were obtained by numerical simulation. Increasing the volume of fluid in the well-altered dimensional but not dimensionless shear metrics. Adding a central cylinder to the base of the well-forced fluid to flow in a square toroidal channel and reduced multidirectionality. Conversely, suspending a cylinder above the base of the well made the flow highly multidirectional. Increasing viscosity in the latter model increased the magnitude of dimensional but not dimensionless metrics. Finally, tilting the well changed the patterns of different wall shear stressmetrics in different ways. Collectively, these methods allow similar flows over most of the cells cultured and/or allow the separation of different shear metrics. A combination of the methods overcomes the limitations of the baseline model.

Determination of the ability of matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify high-biofilm-producing strains.

The traditional method for assessing the capacity of a microorganism to produce biofilm is generally a static in vitro model in a multi-well plate using the crystal violet (CV) binding assay, which takes 96 h. Furthermore, while the method is simple to perform, its reproducibility is poor. We evaluated whether matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) could make it possible to differentiate between high-and low-biofilm-producing microorganisms on 24-h cultures of Staphylococcus aureus and Candida albicans. We included 157 strains of S. aureus and 91 strains of C. albicans obtained from the blood cultures of patients with bacteremia/candidemia. We tested biofilm production using the CV binding assay as the gold standard to classify strains as low or high biofilm producers. We then applied MALDI-TOF MS to create a machine learning-based predictive model using 40 strains of S. aureus and C. albicans, each with extreme absorbance values, and validated this approach with the remaining 117 and 51 strains using the random forest algorithm and the support vector machine algorithm, respectively. Overall, 81.2% of the S. aureus strains (95/117) and 74.5% of the C. albicans strains (38/51) used for validation were correctly categorized, respectively, as low and high-biofilm-producing. Classification based on MALDI-TOF MS protein spectra enables us to predict acceptable information about the capacity of 24-h cultures of S. aureus and C. albicans to form biofilm.

Optimization of Inclusion Body Formation and Purification in Multi-well Plates.

Heterologous expression has long been used for the efficient production of proteins and enzymes as it offers significant advantages over purification of proteins from their native organisms. When first established, great efforts have been made to heterologously express proteins with high yields in the soluble fraction, hence, avoiding protein aggregation. In recent decades, however, it has been shown that the formation of aggregates (inclusion bodies; IBs) can be beneficial. To recover active protein, however, proteins should have been refolded from IBs after purification. The discovery that IBs themselves can also be active has revolutionized the entire protein production field. Therefore, several approaches have been described to generate catalytically active IBs during heterologous expression. Since several extrinsic and intrinsic factors such as protein structure and toxicity, pH and temperature of expression, and the used media might influence the formation of IBs, it is time and material consuming to use shake flask to examine and optimize different expression conditions. However, by using multi-well plates, it is possible to rapidly develop an efficient protocol for the expression of catalytically active IBs in a rational approach. The presented protocol was used for the heterologous expression of a 5'-adenosine monophosphate phosphorylase which forms catalytically active aggregates during expression in E. coli.

Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing.

Drug testing is a vital step in the identification of the potential efficacy of any new/existing drug and/or combinations of drugs. The conventional methods of testing the efficacy of new drugs using multiwell plates are time consuming and prone to evaporation loss and manual error. Microfluidic devices with automated generation of concentration gradients provide a promising alternative. The implementation of such microfluidic devices is still limited owing to the additional expertise and facilities required to fabricate and run these devices. Conventional microfluidic devices also need pumps, tubing, valves, and other accessories, making them bulky and non-portable. To address these problems, we have developed a method for fabricating microfluidic structures using a nonconventional technique by exploiting the Saffman-Taylor instability in lifted Hele-Shaw cells. Multi-channel structure molds with varying dimensions were fabricated by shaping ceramic polymer slurry and retaining the shape. Further using the mold thus made, polydimethyl siloxane (PDMS) devices offering static, stable, diffusion-based gradients were casted using soft lithography. We have demonstrated with COMSOL simulation, as well as using fluorescein isothiocyanate (FITC), a fluorescent dye, that the concentration gradient can be generated in this device, which remains stable for at least 5 days. Using this multichannel device, in vitro drug efficacy was validated with two drugs namely, temozolomide (TMZ) and curcumin, one FDA approved and one under research, on glioblastoma cells (U87MG). The resulting IC50 values were consistent with those reported in the literature. We have also demonstrated the possibility of conducting molecular assays post-drug testing in the device by microtubule staining after curcumin treatment on cervical cancer cells (HeLa). In summary, we have demonstrated a i) user-friendly, ii) portable, static drug testing platform that iii) does not require further accessories and can create iv) a stable gradient for a long duration. Such a device can reduce the time, manual errors, fabrication and running expenditure, and resources needed to a great extent in drug testing.

High-Throughput Expression of Inclusion Bodies on an Automated Platform.

In bioprocesses, which target the production of recombinant proteins as inclusion bodies, the upstream process has a decisive influence on the downstream operations, especially regarding cell disruption, inclusion body purity and composition, and refolding yield. Therefore, optimization of the processes in fed-batch mode is a major issue, and screening for strains and process conditions are performed in highly labor, time and cost intensive shake flasks or multiwell plates. Thus, high-throughput experiments performed similar to the industrial operating conditions offer a possibility to develop efficient and robust upstream processes. We present here an automated platform for Escherichia coli fed-batch cultivations in parallelized minibioreactors. The platform allows execution of experiments under multiple conditions while allowing for real-time monitoring of critical process parameters and a controlled fermentation environment. By this, the main factors that affect yields and quality of inclusion bodies can be investigated, speeding up the development process significantly.

Development of a robust and quantitative high-throughput screening method for assessing phenotypic variation in large Neisseria meningitidis isolate collections

Genome-wide association studies are a powerful approach for identifying determinants of disease. For infectious diseases, high throughput assays are required for measuring the variance in multiple virulence-related phenotypes of large bacterial isolate collections and for association of this phenotypic variance with genotype. The primary limiting factors are cost, effectiveness and a standardized inoculum. A method was developed to create an inoculum array of multiple isolates that could be used for a series of high-throughput multi-isolate phenotypic investigations in a laboratory setting. A key starting point was the standardisation of the inoculum by production of identical batches of each isolate from cells grown to mid-log phase. Cultures with pre-determined optical densities were aliquoted in set patterns into multiple multi-well plates containing 50% glycerol and stored at -80 °C. Prior to a specific assay, an inoculum plate was defrosted and subjected to a brief period of incubation. Control strains can be placed on each plate in order to control for intra-assay variability. A high throughput screen is described in detail for quantification of biofilm formation. This example utilised the crystal violet staining method and multi-assay stock plates containing 16 meningococcal isolates.•Multi-assay stock plate of exponentially growing isolates is cost-effective and simple to implement in a laboratory setting.•This method would predict realistic standard deviations for multiple isolates in phenotypic assays and generate data for performance of power calculations for genotyping.•This method has the potential to identify both known and unknown genetic determinants of phenotypic variability for each tested isolate when paired with genetic analysis of whole genome sequencing data.

Gene Expression Analysis by Quantitative Real-Time PCR for Floral Tissues.

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR) is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental set-up used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler® 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment.