multiwell plates
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2021 ◽  
Vol 12 ◽  
Author(s):  
Caterina Credi ◽  
Valentina Balducci ◽  
U. Munagala ◽  
C. Cianca ◽  
S. Bigiarini ◽  
...  

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective Kv11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations.


2021 ◽  
Author(s):  
Jesus Hernandez ◽  
Kevin D. Ross ◽  
Bruce A. Hamilton

The yeast two-hybrid (Y2H) assay has long been used to identify new protein-protein interaction pairs and to compare relative interaction strengths. Traditional Y2H formats may be limited, however, by use of constitutive strong promoters if expressed proteins have toxic effects or post-transcriptional expression differences in yeast among a comparison group. As a step toward more quantitative Y2H assays, we modified a common vector to use an inducible CUP1 promoter, which showed quantitative induction of several "bait" proteins with increasing copper concentration. Using mouse Nxf1 (homologous to yeast Mex67p) as a model bait, copper titration achieved levels that bracket levels obtained with the constitutive ADH1 promoter. Using a liquid growth assay for an auxotrophic reporter in multiwell plates allowed log-phase growth rate to be used as a measure of interaction strength. These data demonstrate the potential for quantitative comparisons of protein-protein interactions using the Y2H system.


2021 ◽  
Vol 12 ◽  
Author(s):  
Davide Dal Bo ◽  
Leonardo Magneschi ◽  
Mariette Bedhomme ◽  
Elodie Billey ◽  
Etienne Deragon ◽  
...  

Algae belonging to the Microchloropsis genus are promising organisms for biotech purposes, being able to accumulate large amounts of lipid reserves. These organisms adapt to different trophic conditions, thriving in strict photoautotrophic conditions, as well as in the concomitant presence of light plus reduced external carbon as energy sources (mixotrophy). In this work, we investigated the mixotrophic responses of Microchloropsis gaditana (formerly Nannochloropsis gaditana). Using the Biolog growth test, in which cells are loaded into multiwell plates coated with different organic compounds, we could not find a suitable substrate for Microchloropsis mixotrophy. By contrast, addition of the Lysogeny broth (LB) to the inorganic growth medium had a benefit on growth, enhancing respiratory activity at the expense of photosynthetic performances. To further dissect the role of respiration in Microchloropsis mixotrophy, we focused on the mitochondrial alternative oxidase (AOX), a protein involved in energy management in other algae prospering in mixotrophy. Knocking-out the AOX1 gene by transcription activator-like effector nuclease (TALE-N) led to the loss of capacity to implement growth upon addition of LB supporting the hypothesis that the effect of this medium was related to a provision of reduced carbon. We conclude that mixotrophic growth in Microchloropsis is dominated by respiratory rather than by photosynthetic energetic metabolism and discuss the possible reasons for this behavior in relationship with fatty acid breakdown via β-oxidation in this oleaginous alga.


2021 ◽  
Author(s):  
Ida Barlow ◽  
Luigi Feriani ◽  
Eleni Minga ◽  
Adam McDermott-Rouse ◽  
Thomas O'Brien ◽  
...  

Tracking small laboratory animals such as flies, fish, and worms is used for phenotyping in neuroscience, genetics, disease modelling, and drug discovery. Current imaging systems are limited either in spatial resolution or throughput. A system capable of imaging a large number of animals with sufficient resolution to estimate their pose would enable a new class of experiments where detailed behavioural differences are quantified but at a scale where hundreds of treatments can be tested simultaneously. Here we report a new imaging system consisting of an array of six 12-megapixel cameras that can simultaneously record from all the wells of a 96-well plate with a resolution of 80 pixels/mm at 25 frames per second. We show that this resolution is sufficient to estimate the pose of nematode worms including head identification and to extract high-dimensional phenotypic fingerprints. We illustrate the potential application of the system in three domains with a study of behavioural variability across wild isolates, experiments on the escape response and sensitisation of worms to repeated blue light stimulation, and the utility of this stimulus for phenotyping disease models. Because the system is compatible with standard multiwell plates, it makes computational ethological approaches accessible in existing high-throughput pipelines and greatly increases the scale of possible phenotypic screening experiments in C. elegans.


2020 ◽  
Vol 26 (1) ◽  
pp. 32-43
Author(s):  
Alessandra Prinelli ◽  
Catarina Silva-Almeida ◽  
Sisely Parks ◽  
Anna Pasotti ◽  
Aikaterini Telopoulou ◽  
...  

Cell-based assays performed in multiwell plates are utilized in basic and translational research in a variety of cell models. The assembly of these multiwell platforms and their use is often laboratory specific, preventing the standardization of methods and the comparison of outputs across different analytical sites. Moreover, when cell models are based on primary cells with specialized culture requirements, including three-dimensional (3D) cell culture, their complexity and the need for manipulation by experienced operators can add significant cost and introduce long lead times to analysis, both of which are undesirable in any preclinical situation. To address this issue, we explored adaptations of cryopreservation technology that allow cells to be cryopreserved in-plate, ready for use in analysis, and have developed a method applicable to cells from different origins and different culture formats. Here we describe the application of this technology to conventional two-dimensional (2D) monolayers of human mesenchymal stem cells (MSCs) and human macrophages derived from primary monocytes, and to 3D cultures of hepatic organoids, colon organoids, and colon tumor organoids, each presented for cryopreservation in their obligate extracellular matrix. We demonstrated that cell viability, cell physiology, and cytotoxic sensitivity were maintained after cryopreservation, such that the models offer the means to uncouple model assembly from analytical use and to standardize cell models in product form for distribution to end users.


2020 ◽  
Vol 16 ◽  
pp. 2007-2016 ◽  
Author(s):  
Rémi Martinent ◽  
Javier López-Andarias ◽  
Dimitri Moreau ◽  
Yangyang Cheng ◽  
Naomi Sakai ◽  
...  

Recent progress with chemistry tools to deliver into living cells has seen a shift of attention from counterion-mediated uptake of cell-penetrating peptides (CPPs) and their mimics, particularly the Schmuck cation, toward thiol-mediated uptake with cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs), here exemplified by asparagusic acid. A persistent challenge in this evolution is the simultaneous and quantitative detection of cytosolic delivery and cytotoxicity in a high-throughput format. Here, we show that the combination of the HaloTag-based chloroalkane penetration assay (CAPA) with automated high-content (HC) microscopy can satisfy this need. The automated imaging of thousands of cells per condition in multiwell plates allows us to obtain quantitative data on not only the fluorescence intensity but also on the localization in a very short time. Quantitative and statistically relevant results can be obtained from dose–response curves of the targeted delivery to selected cells and the cytotoxicity in the same experiment, even with poorly optimized cellular systems.


Author(s):  
Yifaat Betzalel ◽  
Yoram Gerchman ◽  
Vered Cohen-Yaniv ◽  
Hadas Mamane

2020 ◽  
Author(s):  
Hanjun Henry Hwang ◽  
Shangting You ◽  
Xuanyi Ma ◽  
Leilani Kwe ◽  
Grace Victorine ◽  
...  

2020 ◽  
Vol 1106 ◽  
pp. 79-87
Author(s):  
Emir Yasun ◽  
Baptiste Neff ◽  
Travis Trusty ◽  
Ljuboslav Boskic ◽  
Igor Mezić

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