Long-Term Imaging of Calcium Dynamics Using Genetically Encoded Calcium Indicators and Automatic Tracking of Cultured Cells

Razlivanov, I. N.; Liew, Teresa; Moore, Eira Watts; Al-Kathiri, Alaa; Bartram, Tayma; Kuvshinov, Dmitriy; Nikolaev, Anton

doi:10.2144/btn-2018-0024

Cited by 4 publications

(4 citation statements)

References 10 publications

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“…Although the use of nifedipine assumes clinical significance in the context of ischemia, it should be noted that nifedipine is known to lower blood pressure at a certain dose range, 50 which can, in turn, reduce cerebral perfusion pressure, resulting in reduced O 2 delivery to the tissues. Therefore, the outcome of nifedipine use will be a combination of its effect on the preservation of cell viability and the lowering of cerebral perfusion pressure.…”

Section: Discussionmentioning

confidence: 99%

“…Although the current work is based on a short-term study, the long-term effect of drug testing could provide better applicability to this model. In the future, the use of genetically encoded sensors can be used for long-term Ca 2+ imaging. The other limitation of the study is the use of continuous excitation with lasers that may provide phototoxicity to the live cells.…”

Section: Discussionmentioning

confidence: 99%

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Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock

Dhyani

Kumar

Manne

et al. 2023

ACS Chem. Neurosci.

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Real-time three-dimensional (3-D) imaging is crucial for quantifying correlations among various molecules under acute ischemic stroke. Insights into such correlations may be decisive in selecting molecules capable of providing a protective effect within a shorter period. The major bottleneck is maintaining the cultures under severely hypoxic conditions while simultaneously 3-D imaging intracellular organelles with a microscope. Moreover, comparing the protective effect of drugs and reoxygenation remains challenging. To address this, we propose a novel workflow for the induction of gas-environment-based hypoxia in the HMC-3 cells along with 3-D imaging using laser-scanning-confocal microscopy. The imaging framework is complemented with a pipeline for quantifying time-lapse videos and cell-state classification. First, we show an imaging-based assessment of the in vitro model for hypoxia using a steep gradient in O 2 with time. Second, we demonstrate the correlation between mitochondrial superoxide production and cytosolic calcium under acute hypoxia. We then test the efficacy of an L-type calcium channel blocker, compare the results with reoxygenation, and show that the blocker alleviates hypoxic conditions in terms of cytosolic calcium and viability within an acute window of one hour. Furthermore, we show that the drug reduces the expression of oxidative stress markers (HIF1A and OXR1) within the same time window. In the future, this model can also be used to investigate drug toxicity and efficacy under ischemic conditions.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock

Dhyani

Kumar

Manne

et al. 2023

ACS Chem. Neurosci.

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“…It is low cost (£400-600 without and £2,500 with the fluorescent microscope), is fully customizable, and allows for up to 96 or 384 experiments to be performed, sequentially or, if experiments do not require a high sampling rate (e.g., 1 frame per minute or more), simultaneously. The platform is equipped with a 1-channel epifluorescent microscope head, which can be used to image dynamic fluorescent reporters (e.g., GCaMP and synthetic calcium dyes; Razlivanov et al, 2018) and/or samples labeled with fluorescent antibodies or dyes. We demonstrate how our system allows generating cell-biological data rapidly, under tightly controlled and reproducible experimental conditions, and at large scale.…”

Section: Introductionmentioning

confidence: 99%

An Open-Source Framework for Automated High-Throughput Cell Biology Experiments

Katunin

Zhou

Shehata

et al. 2021

Front. Cell Dev. Biol.

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Modern data analysis methods, such as optimization algorithms or deep learning have been successfully applied to a number of biotechnological and medical questions. For these methods to be efficient, a large number of high-quality and reproducible experiments needs to be conducted, requiring a high degree of automation. Here, we present an open-source hardware and low-cost framework that allows for automatic high-throughput generation of large amounts of cell biology data. Our design consists of an epifluorescent microscope with automated XY stage for moving a multiwell plate containing cells and a perfusion manifold allowing programmed application of up to eight different solutions. Our system is very flexible and can be adapted easily for individual experimental needs. To demonstrate the utility of the system, we have used it to perform high-throughput Ca2+ imaging and large-scale fluorescent labeling experiments.

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“…Its small epifluorescent microscope can be used to perform imaging of fluorescent reporters (e.g. GCaMP and synthetic calcium dyes, [11]) or samples labelled with fluorescent antibodies or dyes. The proposed experimental framework will make generation of biological data faster, more accurate and on a larger scale.…”

Section: Introductionmentioning

confidence: 99%

An open-source experimental framework for automation of high-throughput cell biology experiments

Katunin

Cadby

Nikolaev

2020

Preprint

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AbstractModern data analysis methods, such as deep learning, have been successfully applied to a number of biological and medical questions. For these methods to be efficient, a large number of high quality experiments need to be conducted, which requires a high degree of automation. Here we report an open-source hardware that allows for automatic high-throughput generation of large amounts of biological data. The hardware consists of an automatic XY-stage for moving multiwell plates containing growing cells; a perfusion manifold allowing application (perfusion) of up to 8 different solutions; and a small epifluorescent microscope. It is extremely cheap (£300 without and £2500 with a fluorescent microscope) and can be quickly customised for individual experimental needs.Key points- We present an open source framework for automation of cell biology experiments- The framework consists of an XY platform, application of up to 8 solutions and a small epifluorescent microscope- Very cheap (£300 without a fluorescent microscope and £2500 with a fluorescent microscope), customisable, 3D printable- Can be used in a variety of biological applications such as imaging of fluorescent reporters, optimisation of treatment conditions and immuno-labelling

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Long-Term Imaging of Calcium Dynamics Using Genetically Encoded Calcium Indicators and Automatic Tracking of Cultured Cells

Cited by 4 publications

References 10 publications

Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock

Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock

An Open-Source Framework for Automated High-Throughput Cell Biology Experiments

An open-source experimental framework for automation of high-throughput cell biology experiments

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