Multiwell Plates Research Articles

The amplification of alpha‐synuclein amyloid fibrils is suppressed under fully quiescent conditions

Seed amplification assays (SAAs) are a promising avenue for the early diagnosis of neurodegenerative diseases. However, when amplifying fibrils from patient‐derived samples in multiwell plates, it is currently highly challenging to accurately quantify the aggregates. It is therefore desirable to transfer such assays into a digital format in microemulsion droplets to enable direct quantification of aggregate numbers. To achieve transfer from conventional plate‐based to the microfluidic digital format, effective seed amplification needs to be achieved inside the microdroplets. Therefore, we establish a new set of assay conditions that enable highly efficient seed amplification in plates without any shaking. However, the same set of conditions displayed a very different behavior upon transfer to a microfluidic platform where no amplification was observed. We demonstrate that this is caused by the suppression of all secondary processes that could amplify the seeds in the complete absence of mechanical perturbations inside the microdroplets. We further show that the amplification inside droplets can be achieved by subjecting the microemulsions to high‐frequency vibrations using a piezo device. Taken together, our results provide novel insights into the physical requirements of alpha‐synuclein seed amplification and demonstrate a pathway towards the development of effective digital SAAs.

The amplification of alpha-synuclein amyloid fibrils is suppressed under fully quiescent conditions.

Seed amplification assays (SAAs) are a promising avenue for the early diagnosis of neurodegenerative diseases. However, when amplifying fibrils from patient-derived samples in multiwell plates, it is currently highly challenging to accurately quantify the aggregates. It is therefore desirable to transfer such assays into a digital format in microemulsion droplets to enable direct quantification of aggregate numbers. To achieve transfer from conventional plate-based to the microfluidic digital format, effective seed amplification needs to be achieved inside the microdroplets. Therefore, we establish a new set of assay conditions that enable highly efficient seed amplification in plates without any shaking. However, the same set of conditions displayed a very different behavior upon transfer to a microfluidic platform where no amplification was observed. We demonstrate that this is caused by the suppression of all secondary processes that could amplify the seeds in the complete absence of mechanical perturbations inside the microdroplets. We further show that the amplification inside droplets can be achieved by subjecting the microemulsions to high-frequency vibrations using a piezo device. Taken together, our results provide novel insights into the physical requirements of alpha-synuclein seed amplification and demonstrate a pathway towards the development of effective digital SAAs.

AutoMEA: machine learning-based burst detection for multi-electrode array datasets

Neuronal activity in the highly organized networks of the central nervous system is the vital basis for various functional processes, such as perception, motor control, and cognition. Understanding interneuronal connectivity and how activity is regulated in the neuronal circuits is crucial for interpreting how the brain works. Multi-electrode arrays (MEAs) are particularly useful for studying the dynamics of neuronal network activity and their development as they allow for real-time, high-throughput measurements of neural activity. At present, the key challenge in the utilization of MEA data is the sheer complexity of the measured datasets. Available software offers semi-automated analysis for a fixed set of parameters that allow for the definition of spikes, bursts and network bursts. However, this analysis remains time-consuming, user-biased, and limited by pre-defined parameters. Here, we present autoMEA, software for machine learning-based automated burst detection in MEA datasets. We exemplify autoMEA efficacy on neuronal network activity of primary hippocampal neurons from wild-type mice monitored using 24-well multi-well MEA plates. To validate and benchmark the software, we showcase its application using wild-type neuronal networks and two different neuronal networks modeling neurodevelopmental disorders to assess network phenotype detection. Detection of network characteristics typically reported in literature, such as synchronicity and rhythmicity, could be accurately detected compared to manual analysis using the autoMEA software. Additionally, autoMEA could detect reverberations, a more complex burst dynamic present in hippocampal cultures. Furthermore, autoMEA burst detection was sufficiently sensitive to detect changes in the synchronicity and rhythmicity of networks modeling neurodevelopmental disorders as well as detecting changes in their network burst dynamics. Thus, we show that autoMEA reliably analyses neural networks measured with the multi-well MEA setup with the precision and accuracy compared to that of a human expert.

LabEmbryoCam: An opensource phenotyping system for developing aquatic animals

Phenomics is the acquisition of high-dimensional data on an individual-wide scale and is proving transformational in areas of biological research related to human health including medicine and the crop sciences. However, more broadly, a lack of accessible transferrable technologies and research approaches is significantly hindering the uptake of phenomics, in contrast to molecular-omics for which transferrable technologies have been a significant enabler. Aquatic embryos are natural models for phenomics, due to their small size, taxonomic diversity, ecological relevance, and high levels of temporal, spatial and functional change. Here, we present LabEmbryoCam, an autonomous phenotyping platform for timelapse imaging of developing aquatic embryos cultured in a multiwell plate format, and while optimised for embryos, the instrument is extremely versatile. The LabEmbryoCam capitalises on 3D printing, single board computers, consumer electronics and stepper motor enabled motion. These provide autonomous X, Y and Z motion, a web application streamlined for rapid setup of experiments, user email notifications and a humidification chamber to reduce evaporation over prolonged acquisitions. Downstream analyses are provided, enabling automated embryo segmentation, heartbeat detection, motion tracking, and energy proxy trait (EPT) measurement. LabEmbryoCam is a scalable, and flexible laboratory instrument, that leverages embryonic and early life stage organisms to tackle key global challenges including biological sensitivity assessment, toxicological screening, but also to support broader engagement with the earliest stages of life.

Enhancing Doxorubicin Detection: Multiphase Electroextraction for Efficient and Affordable UHPLC-DAD Analysis in Saliva.

Attesting optimal drug concentrations in biological fluids is crucial to ensure precise dosage adjustment, to guarantee therapy adherence, and to manage side effects in chemotherapy. Accurate drug determination relies on liquid chromatography and advanced detectors, with sample preparation playing a pivotal role, especially in complex matrices such as biological fluids. This study introduces a multiphase electroextraction (MPEE) of doxorubicin (DOX) in saliva by utilizing a paper point, followed by ultra-high-performance liquid chromatography coupled to diode array detection. The extraction time and electric potential were carried out by using the Doehlert optimization approach, whereas the desorption solvent was fine-tuned through the centroid-simplex experimental design. After optimization, DOX and the internal standard were extracted in 35min, utilizing an applied voltage of 300V and a multiwell plate capable of simultaneous extraction of 66 samples. The recovery was 87%-101%, with a linear range between 50 and 500µgL-1 (R2>0.999). The intra- and inter-assay coefficients of variation for precision were <10%, and the limit of detection and limit of quantification were 25 and 50µgL-¹, respectively. When applied to five different fortified saliva samples, there were no statistically significant differences in the detected concentrations. Although the enrichment factor (0.6) was not as high as expected, the other results confirm that the method obtained is suitable for monitoring DOX in this complex matrix and can contribute to further developments in sample preparation using MPEE approaches.

Automated image registration of RGB, hyperspectral and chlorophyll fluorescence imaging data

BackgroundThe early and specific detection of abiotic and biotic stresses, particularly their combinations, is a major challenge for maintaining and increasing plant productivity in sustainable agriculture under changing environmental conditions. Optical imaging techniques enable cost-efficient and non-destructive quantification of plant stress states. Monomodal detection of certain stressors is usually based on non-specific/indirect features and therefore is commonly limited in their cross-specificity to other stressors. The fusion of multi-domain sensor systems can provide more potentially discriminative features for machine learning models and potentially provide synergistic information to increase cross-specificity in plant disease detection when image data are fused at the pixel level.ResultsIn this study, we demonstrate successful multi-modal image registration of RGB, hyperspectral (HSI) and chlorophyll fluorescence (ChlF) kinetics data at the pixel level for high-throughput phenotyping of A. thaliana grown in Multi-well plates and an assay with detached leaf discs of Rosa × hybrida inoculated with the black spot disease-inducing fungus Diplocarpon rosae. Here, we showcase the effects of (i) selection of reference image selection, (ii) different registrations methods and (iii) frame selection on the performance of image registration via affine transform. In addition, we developed a combined approach for registration methods through NCC-based selection for each file, resulting in a robust and accurate approach that sacrifices computational time. Since image data encompass multiple objects, the initial coarse image registration using a global transformation matrix exhibited heterogeneity across different image regions. By employing an additional fine registration on the object-separated image data, we achieved a high overlap ratio. Specifically, for the A. thaliana test set, the overlap ratios (ORConvex) were 98.0 ± 2.3% for RGB-to-ChlF and 96.6 ± 4.2% for HSI-to-ChlF. For the Rosa × hybrida test set, the values were 98.9 ± 0.5% for RGB-to-ChlF and 98.3 ± 1.3% for HSI-to-ChlF.ConclusionThe presented multi-modal imaging pipeline enables high-throughput, high-dimensional phenotyping of different plant species with respect to various biotic or abiotic stressors. This paves the way for in-depth studies investigating the correlative relationships of the multi-domain data or the performance enhancement of machine learning models via multi modal image fusion.

Multi-well plate-based versatile platform for online fabricating alginate hydrogel microspheres and in-situ 3D cell culture

BackgroundHydrogel microspheres with monodisperse and homogeneous dimensions have potential application in the field of three-dimensional (3D) cell culture due to its ability to provide a similar microenvironment. Currently, alginate hydrogel microspheres (AHMs) have received much attention due to the favorable properties of alginate such as biocompatibility, inexpensiveness, nontoxicity, and biodegradability. The fabrication methods of AHMs mainly include extrusion, electrostatic dripping and microfluidic chip techniques. These current methods suffer trade-offs between operational complexity, fabrication cost and practical application. ResultsWe proposed a novel and versatile multi-well plate-based platform for online fabricating AHMs and in-situ 3D cell culture. The AHMs could be easily fabricated based on gravity-driven gelation combined with our recently developed bent-capillary-centrifugal-driven (BCCD) system. Ca-EDTA complex was used as Ca2+ source for crosslinking reaction of the alginate chains. The whole preparation process of AHMs included four steps: emulsification, pre-gelation, spontaneous demulsification and further solidification. The gravity-driven hydrogel microsphere gelation could produce the AHMs with good sphericity (Φ = 0.96) and monodispersity (PDI% = 0.94 %). The rapid drug susceptibility testing and single-cell encapsulation in the AHMs were well demonstrated. It also provided a novel in-situ 3D cell culture strategy, which demonstrated more than 85 % cell viability in practice. SignificanceThe proposed platform avoided the complex and laborious microfabrication. Moreover, cell-encapsulated AHMs could be directly produced in the multi-well plate, which could facilitate the subsequent cultivation and observation. It is expected to be a versatile in-situ 3D cell culture tool in the fields of biomedicine and tissue engineering.

Identification of plasma proteins binding oxidized phospholipids using pull-down proteomics and OxLDL masking assay

Oxidized phospholipids (OxPLs) are increasingly recognized as toxic and proinflammatory mediators, which raises interest in the mechanisms of their detoxification. Circulating OxPLs are bound and neutralized by plasma proteins, including both antibodies and non-immunoglobulin proteins. The latter group of proteins is essentially not investigated because only three OxPC-binding plasma proteins are currently known. The goal of this work was to characterize a broad spectrum of plasma proteins selectively binding OxPLs. Using pull-down-proteomic analysis, we found about 150 non-immunoglobulin proteins preferentially binding oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphatidylcholine (OxPAPC) as compared to non-oxidized PAPC. To test if candidate proteins indeed can form a barrier isolating OxPLs from recognition by other proteins, we applied an immune masking assay. Oxidized LDL (OxLDL) immobilized in multiwell plates was used as a carrier of OxPLs, while mAbs recognizing OxPC or OxPE were used as “detectors” showing if OxPLs on the surface of OxLDL are physically accessible to external binding partners. Using an orthogonal combination of pull-down and masking assays we confirmed that previously described OxPL-binding proteins (non-fractionated IgM, CFH, and Apo-M) indeed can bind to and mask OxPC and OxPE on liposomes and OxLDL. Furthermore, we identified additional plasma proteins selectively binding and masking OxPC including Apo-D, Apo-H, pulmonary surfactant-associated protein B, and antithrombin-III. We hypothesize that in addition to circulating antibodies, multiple non-immunoglobulin plasma proteins can also bind OxPLs and modulate their recognition by innate and adaptive immunity and that an orthogonal combination of pull-down and masking assays is a useful approach for the identification of such proteins.

Development and validation of an in vitro model to study thrombin generation on the surface of catheters in platelet-poor and platelet-rich plasma

IntroductionCoagulation activation on medical devices remains a significant problem as it can lead to dramatic thromboembolic complications. Understanding its poorly described mechanisms and finding optimal pharmacological prevention means is crucial to improve patient safety. MethodsWe developed an in vitro model to study thrombin generation (TG) initiated by the contact of plasma with the surface of catheters. Interventional cardiology catheters were cut into segments and inserted in the bottom of multi-well plates; TG was then measured with the calibrated automated thrombogram (CAT). Model performance (analytical, intra- and inter-individual variability) was investigated and compared with activation of thrombin generation by tissue factor (TF) or contact pathway activator (ellagic acid), in the presence (PRP) and absence (PPP) of platelets. Model response to unfractionated heparin (UFH) was also assessed. ResultsTG was greater when measured in presence of catheter segments, compared to conditions without activators. The analytical variability of the model was good (CV ≤ 5 %), both with PPP and PRP. Intra-individual variability was between 15 and 30 % with PPP and between 10 and 15 % with PRP. Inter-individual variability was between 15 and 30 % with both kinds of plasma samples. The analytical performance of the catheter-initiated TG model was equivalent to that observed when TG was initiated with TF or ellagic acid. Catheter-initiated TG was measurable until 0.1 IU/mL UFH with PPP and until 1.0 IU/mL UFH with PRP, highlighting the crucial requirement of platelets. ConclusionOur model is suitable for studying TG initiated with catheters. Inhibition of TG by UFH is overestimated in the absence of platelets.

Environmentally Controlled Microfluidic System Enabling Immune Cell Flow and Activation in an Endothelialised Skin-On-Chip.

Integration of reconstructed human skin (RhS) into organ-on-chip (OoC) platforms addresses current limitations imposed by static culturing. This innovation, however, is not without challenges. Microfluidic devices, while powerful, often encounter usability, robustness, and gas bubble issues that hinder large-scale high-throughput setups. This study aims to develop a novel re-usable multi-well microfluidic adaptor (MMA) with the objective to provide a flexible tool for biologists implementing complex 3D biological models (e.g., skin) while enabling simultaneous user control over temperature, medium flow, oxigen (O2), nitrogen (N2), and carbon dioxide (CO2) without the need for an incubator. The presented MMA device is designed to be compatible with standard, commercially available 6-well multi-well plates (6MWPs) and 12-well transwells. This MMA-6MWP setup is employed to generate a skin-on-chip (SoC). RhS viability is maintained under flow for three days and their morphology closely resembles that of native human skin. A proof-of-concept study demonstrates the system's potential in toxicology applications by combining endothelialised RhS with flowing immune cells. This dynamic setting activates the monocyte-like MUTZ-3 cells (CD83 and CD86 upregulation) upon topical exposure of RhS to a sensitizer, revealing the MMA-6MWP's unique capabilities compared to static culturing, where such activation is absent.

8660 Bodipy Labeled Vitamin D3 Associates With Lipid Droplets In Adipocytes

Abstract Disclosure: N. Ucar: None. J.T. Deeney: None. R. Pickering, PhD: None. M.T. Kirber: None. T. Fan: None. R. Loo, PhD: Research Investigator; Self; Carbogen Amcis BV. M.F. Holick: Grant Recipient; Self; Carbogen Amcis BV. Research Investigator; Self; Carbogen Amcis BV. Obese patients are often found to be deficient in vitamin D, requiring higher levels of supplementation than normal weight subjects. While this has been attributed to vitamin D3‘s lipid solubility and accumulation in fat tissue, little is known about how vitamin D3 enters adipocytes and associates with the intracellular lipid droplet. The goal of this study was to investigate this association using a novel fluorescently labelled vitamin D3. Bodipy, 4,4-difluoro-1,3,5, 7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503), was covalently linked to vitamin D3 and its structure was confirmed by NMR and mass spectroscopy. Murine 3T3L1 and primary adipocytes were used for our study. Mouse 3T3L1 preadipocytes were cultured in multiwell plates or glass bottom dishes in high glucose DMEM media and differentiated for 7-14 days. Primary adipocytes were obtained by collagenase digestion after surgical extraction of adipose tissue from male/female C57BL/6J mice and attached to glass bottom dishes. The cultured adipocytes were exposed to 10 microM BODIPY labelled Vitamin D3 (vitD-B). BODIPY was added to cultured cells and served as the control. Cells were imaged at various times on a wide-field epifluorescence microscope and analysed with NIH ImageJ software. VitD-B adipocyte uptake and accumulation in lipid droplets was observed over 24 hrs. Isolated lipid droplets were also incubated with vitD-B and BODIPY. VitD-B in mature 3T3L1 cells was observed in lipid droplets after 1h incubation and increased fluorescent intensity was observed over the next 24 hrs. BODIPY was observed in the lipid droplets after 15-20 mins. As a negative control, non-differentiated 3T3L1 cells did not exhibit staining by BODIPY even after 24-hrs. When vitD-B was added to primary cultured adipocytes, it appeared in the adipocytes within 1-hr. Fluorescence intensity of primary adipocytes was increased 1.2-fold at 8 hrs and 5.7-fold at 24 hrs compared to 1 hr. For comparison the positive control treated with BODIPY exhibited rapid fluorescence uptake into primary adipocytes that was increased 29-fold higher than that of vitD-B at 1 hr. VitD-B and BODIPY both demonstrated rapid uptake into isolated lipid droplets (less than 2 min) based on fluorescence microscopy.These results hold promising implications for understanding of the interaction between vitamin D and intracellular lipid droplets. Keywords: Adipocyte, Vitamin D3, BODIPY, adipocytes, lipid droplet, fluorescent labelled vitamin D3 Presentation: 6/2/2024

Automated multimodal imaging of Caenorhabditis elegans behavior in multi-well plates.

Assays of behavior in model organisms play an important role in genetic screens, drug testing, and the elucidation of gene-behavior relationships. We have developed an automated, high-throughput imaging and analysis method for assaying behaviors of the nematode C. elegans. We use high-resolution optical imaging to longitudinally record the behaviors of 96 animals at a time in multi-well plates, and computer vision software to quantify the animals' locomotor activity, behavioral states, and egg laying events. To demonstrate the capabilities of our system we used it to examine the role of serotonin in C. elegans behavior. We found that egg-laying events are preceded by a period of reduced locomotion, and that this decline in movement requires serotonin signaling. In addition, we identified novel roles of serotonin receptors SER-1 and SER-7 in regulating the effects of serotonin on egg laying across roaming, dwelling, and quiescent locomotor states. Our system will be useful for performing genetic or chemical screens for modulators of behavior.

Passive Droplet Microfluidic Platform for High-Throughput Screening of Microbial Proteolytic Activity.

Traditional bacterial isolation methods are often costly, have limited throughput, and may not accurately reflect the true microbial community composition. Consequently, identifying rare or slow-growing taxa becomes challenging. Over the past decade, a new approach has been proposed to replace traditional flasks or multiwell plates with ultrahigh-throughput droplet microfluidic screening assays. In this study, we present a novel passive droplet-based method designed for isolating proteolytic microorganisms, which are crucial in various biotechnology industries. Following the encapsulation of single cells in gelatin microgel compartments and their subsequent clonal cultivation, microcultures are passively sorted at high throughput based on the deformability of droplets. Our novel chip design offers a 50-fold improvement in throughput compared to a previously developed deformability-based droplet sorter. This method expands an array of droplet-based microbial enrichment assays and significantly reduces the time and resources required to isolate proteolytic bacteria strains.

Photodynamic Therapy Using a Rose-Bengal Photosensitizer for Hepatocellular Carcinoma Treatment: Proposition for a Novel Green LED-Based Device for In Vitro Investigation.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Despite new treatments, the HCC rate remains important, making it necessary to develop novel therapeutic strategies. Photodynamic therapy (PDT) using a Rose-Bengal (RB) photosensitizer (RB-PDT) could be a promising approach for liver tumor treatment. However, the lack of standardization in preclinical research and the diversity of illumination parameters used make comparison difficult across studies. This work presents and characterizes a novel illumination device based on one green light-emitting diode (CELL-LED-550/3) dedicated to an in vitro RB-PDT. The device was demonstrated to deliver a low average irradiance of 0.62 mW/cm2 over the 96 wells of a multi-well plate. Thermal characterization showed that illumination does not cause cell heating and can be performed inside an incubator, allowing a more rigorous assessment of cell viability after PDT. An in vitro cytotoxic study of the RB-PDT on an HCC cell line (HepG2) demonstrated that RB-PDT induces a significant decrease in cell viability: almost all the cells died after a light dose irradiation of 0.3 J/cm2 using 75 µM of RB (<10% of viability). In conclusion, the RB-PDT could be a therapeutic option to treat unresectable liver lesions and subclinical disease remaining in the post-resection tumor surgical margin.

99 The Effects of β-caryophyllene on butyrate utilization and metabolism in Caco 2 cells

Abstract Butyrate is important for regulating energy status and cellular metabolism. Beta-caryophyllene (BCP) is a plant compound which may exert potentially beneficial effects on intestinal epithelial cells, such as barrier integrity and modulation of nutrient utilization. The goal of this preliminary study was to investigate the effects of BCP on butyrate utilization and metabolism in intestinal epithelial cells. Caco 2 cells were used as a model for the intestinal barrier. Cells were seeded in multi-well plates with hanging inserts (n = 4) and grown for 18 d. On d 19, 1 of 4 treatments was added to triplicate wells per plate. Treatments included vehicle control (VC), 40 µM BCP, 2 mM butyrate (BTR), and butyrate plus BCP (BB). Treatments were added to the apical side and incubated for 24 and 48 h. Transepithelial electrical resistance (TEER; ohms/cm2) readings and supernatant samples from both the apical and basolateral sides of the cell layer were taken at each time point, and butyrate and β- hydroxybutyrate (BHB) concentrations measured. Butyrate utilization was calculated as absolute nmol lost. Statistical analyses were performed using lm and emmeans packages of R, with treatment, time and side (of cell layer) as fixed effects. All wells started with the same statistical TEER values on h 0. After 24 and 48 h BCP showed no changes in TEER (P &amp;gt; 0.05). Compared with VC, the mean TEER for BTR and BB was greater at 24 h (579 vs 878 and 1,008 ± 35, respectively; P &amp;lt; 0.001). After 48 h, all groups returned to baseline, except BB which had a greater TEER (883 ± 35; P &amp;lt; 0.001) compared with other treatments. After 24 and 48 h BCP showed no changes for both sides of the cell layer (P &amp;gt; 0.05). Overall, absolute BHB concentrations increased from 24 to 48 h in all treatments (P &amp;lt; 0.05). Production of BHB in each of the VC and BC groups was less than 15 nmol at each time point. For BB and BTR groups, more nmol BHB was detected on the basolateral side (31.9 and 22.2 ± 1.3) vs apical (14.0 and 10.4 ± 1.3, respectively; P &amp;lt; 0.001) after 24 h. After 48 h, BHB was also greater on the basolateral side (58.2 and 45.2 ± 1.3 nmol for BB and BUT, respectively) vs apical side (30.2 and 24.0 ± 1.3 nmol; P &amp;lt; 0.001). When looking at BTR utilization, the BB treatment had greater butyrate disappearance than BTR in 24 h (254 vs 169 ± 12.6 nmol; P = 0.0005) and 48h (707 vs 573 ± 12.6 nmol; P &amp;lt; 0.0001). BCP may promote the metabolism of butyrate by intestinal epithelial cells. More research is needed to understand the mechanism of how BCP increases cellular butyrate utilization.

Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry

Abstract Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions. Yet, we lack practical adaptations to fully harness the quantitative and high-throughput capabilities of most cytometers for many organisms. Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. In proof-of-principle experiments, we show that DNA content increase in S-phase correlates with de novo DNA synthesis and can be predicted by the presence of the replicative helicase MCM2-7 on genomic DNA.

Automated Image-Based Fluorescence Screening of Mitochondrial Membrane Potential in Daphnia magna: An Advanced Ecotoxicological Testing Tool.

This study demonstrated the strengths of in vivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30 min staining with 2 μM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using an automated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2 h exposure to environmentally relevant concentrations and predicted organism death was observed after 24 h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

Automated multimodal imaging of Caenorhabditis elegans behavior in multi-well plates.

Assays of behavior in model organisms play an important role in genetic screens, drug testing, and the elucidation of gene-behavior relationships. We have developed an automated, high-throughput imaging and analysis method for assaying behaviors of the nematode C. elegans . We use high-resolution optical imaging to longitudinally record the behaviors of 96 animals at a time in multi-well plates, and computer vision software to quantify the animals' locomotor activity, behavioral states, and egg laying events. To demonstrate the capabilities of our system we used it to examine the role of serotonin in C. elegans behavior. We found that egg-laying events are preceded by a period of reduced locomotion, and that this decline in movement requires serotonin signaling. In addition, we identified novel roles of serotonin receptors SER-1 and SER-7 in regulating the effects of serotonin on egg laying across roaming, dwelling, and quiescent locomotor states. Our system will be useful for performing genetic or chemical screens for modulators of behavior.

A rapid and facile immunoassay for C-reactive protein using PDMS-based digital magnetofluidics

BackgroundC-reactive protein has been reported as a biomarker of inflammation caused by acute injury, infection or tissue damage and also a prediction marker of cardiovascular diseases. Commonly, the gold standard for the detection of CRP is enzyme-linked immunosorbent assays (ELISAs). Normally, traditional immunoassays in multiwell plates typically suffer from prolonged assay time due to slow mass transport controlled by diffusion. Herein, a PDMS based magnetofluidic approach has been applied for a rapid and facile immunoassay using a sandwich enzyme-linked immunosorbent assay (ELISA) for the analysis of CRP. ResultsDue to the superhydrophobic PDMS, droplets of reagent and sample solutions were obtained when pipetting all solutions onto the PDMS substrate. These droplets were individually controlled by an external magnet to perform the assays. Magnetic beads immobilized with a capture antibody were not only used for immunomagnetic separation (IMS) of the captured CRP from the sample matrix, but also used as a carrier for droplet movement on the magnetofluidic device, expediting the immunoassay procedure, especially washing steps. The immunoassay of CRP was successfully performed within 1 h with a limit of detection of 0.015 mg L−1 in the concentration range of 0.1–10 mg L−1. The recovery percentages of CRP spiked in human serum were found in the range of 90–114 % with %RSD of less than 5 %, indicating acceptable accuracy and precision. SignificanceBy individually controlling the droplet movement using an external magnet, all steps of immunoassays were simply and rapidly performed. In addition, the microfluidic format allows for small volumes of reagents and samples and rapid assay kinetics. Therefore, the proposed magnetofluidic approach has shown its potential of becoming a rapid, facile and cost-effective method to perform traditional immunoassays in a variety of applications. In addition, the proposed approach is also particularly well-suited for analyses/reactions with multiple steps.

In vitro evaluation of the toxicological effects of cooking oil fumes using a self-designed microfluidic chip

Cooking oil fumes (COFs) are widely acknowledged as substantial contributors to indoor air pollution, having detrimental effects on human health. Despite the existence of commercialized in vitro aerosol exposure platforms, assessment risks of aerosol pollutants are primarily evaluated based on multiwell plate experiments by trapping and redissolving aerosols to conduct comprehensive in vitro immersion exposure manner. Therefore, an innovative real-time exposure system for COF aerosol was constructed, featuring a self-designed microfluidic chip as its focal component. The chip was used to assess toxicological effects of in vitro exposure to COF aerosol on cells cultured at the gas–liquid interface. Meanwhile, we used transcriptomics to analyze genes that exhibited differential expression in cells induced by COF aerosol. The findings indicated that the MAPK signaling pathway, known for its involvement in inflammatory response and oxidative stress, played a crucial role in the biological effects induced by COF aerosol. Biomarkers associated with inflammatory response and oxidative stress exhibited corresponding alterations. Furthermore, the concentration of COF aerosol exposure and post-exposure duration exert decisive effects on these biomarkers. Thus, the study suggests that COF can induce oxidative stress and inflammatory response in BEAS-2B cells, potentially exerting a discernible impact on human health.