Name: live-cell imaging of single-cell arrays (lisca) - a versatile technique to quantify cellular kinetics
Uploaded: 2021-03-18T14:30:00
Description: Watch this Scientific Journal Video about Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics at JoVE.com

This work was supported by grants from the German Science Foundation (DFG) to Collaborative Research Center (SFB) 1032. Support by the German Federal Ministry of Education, Research and Technology (BMBF) under the cooperative project 05K2018-2017-06716 Medisoft as well as a grant from the Bayerische Forschungsstiftung are gratefully acknowledged. Anita Reiser was supported by a DFG Fellowship through the Graduate School of Quantitative Biosciences Munich (QBM).

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62025/62025fig02.jpg" /> 
Figure 2: Data acquisition combining single-cell microarrays (A) with scanning time-lapse microscopy (B).&#160;As preparation of the time-lapse experiment, a single-cell array with a 2D micropattern of adhesion squares is prepared (1), followed by cell seeding and the alignment of the cells on the micropattern (2) as well as the connection of a perfusion system to the six-channel slide,...

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Here we described LISCA as a versatile technique to follow cellular kinetics of intracellular fluorescent labels at the single-cell level. In order to perform a successful LISCA experiment, each of the described steps of the protocol section must be established individually and then all steps must be combined. Each of the three major aspects of LISCA feature crucial steps.
Single-cell microarray fabrication 
The quality of the microarray is crucial as the cellular alignment on th...

In recent years, the importance of single-cell experiments has become apparent. Data from single cells allow the investigation of cell-to-cell variability, the resolution of intracellular parameter correlations and the detection of cellular kinetics that remain hidden in ensemble measurements1,2,3. In order to investigate cellular kinetics of thousands of single cells in parallel, new approaches are needed that enable monitoring the cells under standardized conditions over a time period of several hours up to several days followed by a quantitative data analysis 4. Here, we present Live-cell Imaging of Single-Cell Arrays (LISCA), which combines the use of microstructured arrays with time-lapse microscopy and automated image analysis.
Several methods for generating microstructured single-cell arrays have been established and published in literature5,6. Here, we briefly describe Microscale Plasma-Initiated Protein Patterning (&#181;PIPP). A detailed protocol of the single-cell array fabrication using &#181;PIPP is also found in reference7. The use of single-cell arrays enables alignment of thousands of cells on standardized adhesion spots presenting defined microenvironments for each cell and thus reduces a source of experimental variability (Figure 1A). Single-cell arrays are used to monitor the time courses of fluorescent markers purposed to indicate a variety of cellular processes. Long-term microscopy in scanning time-lapse mode allows for monitoring a large area of the single-cell arrays and hence sampling single-cell data in high-throughput over an observation time of several hours or even days. This generates time-line stacks of images from each position of the array (Figure 1B). In order to reduce the large amount of image data and to extract the desired single-cell fluorescence time courses in an efficient way, automated image processing is required that takes advantage of the positioning of cells (Figure 1C).
The challenge of LISCA is to adapt the experimental protocols and computational tools to form a high-throughput assay that generates quantitative and reproducible data of cellular kinetics. In this article we provide a step-by-step description of the individual methods and how they are combined in a LISCA assay. As an example, we discuss the time course of enhanced green fluorescent protein (eGFP) expression after artificial mRNA delivery. eGFP expression following mRNA delivery is described by reaction rate equations modeling translation and degradation of mRNA. Fitting the model function for the time course of eGFP concentration to the LISCA readout of the fluorescence intensity for each individual cell over time yields best estimates of model parameters such as the mRNA degradation rate. As a representative result we discuss the mRNA delivery efficiency of two different lipid-based transfection agents and how their parameter distributions differ.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62025/62025fig01.jpg" /> 
Figure 1: Representation of the LISCA workflow combining (A) micro-patterned single-cell arrays (B) scanning time-lapse microscopy and (C) automated image analysis of recorded image series.&#160;The single-cell arrays consist of a two-dimensional pattern of cell-adhesive squares with a cell-repellent interspace leading to an arrangement of the cells on the micropattern, as can be seen in the phase-contrast image as well as the fluorescence image of eGFP expressing cells (A). The entire microstructured area is imaged in a scanning time-lapse mode repeatedly taking images at a sequence of positions (B). Recorded image series are processed to read out the fluorescence intensity per cell over time (C). Scale bars: 500 &#181;m (A), 200 &#181;m (C).&#160;<a href="https://www.jove.com/files/ftp_upload/62025/62025fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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			<td>Adtech Polymer Engineering PTFE Microtubing&#160;</td>
			<td>Fisher Scientific</td>
			<td>10178071</td>
			<td></td>
		</tr>
		<tr>
			<td>baking oven</td>
			<td>Binder</td>
			<td>9010-0190</td>
			<td></td>
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			<td>CFI Plan Fluor DL 10x</td>
			<td>Nikon</td>
			<td>MRH20100</td>
			<td></td>
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			<td>Desiccator</td>
			<td>Roth</td>
			<td>NX07.1</td>
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			<td>Eclipse Ti-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
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		<tr>
			<td>eGFP mRNA</td>
			<td>Trilink</td>
			<td>L-7601</td>
			<td></td>
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			<td>Female Luer to Tube Connector</td>
			<td>MEDNET</td>
			<td>FTL210-6005</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Yo Proteins</td>
			<td>663</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter set eGFP</td>
			<td>AHF</td>
			<td>F46-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Translucent Platinum-Cured Silicone Tubing</td>
			<td>Fisher Scientific</td>
			<td>11768088</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation Box</td>
			<td>Okolab</td>
			<td>OKO-H201</td>
			<td></td>
		</tr>
		<tr>
			<td>incubator</td>
			<td>Binder</td>
			<td>9040-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 without phenol red</td>
			<td>Thermo Fisher</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Luer</td>
			<td></td>
			<td></td>
			<td>in-house fabricated consisting of teflon</td>
		</tr>
		<tr>
			<td>Male Luer to Tube Connector</td>
			<td>MEDNET</td>
			<td>MTLS210-6005</td>
			<td>alternative to in-house fabricated male luers</td>
		</tr>
		<tr>
			<td>NaCl (5 M)</td>
			<td>Thermo Fisher</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needleless Valve to Male Luer Connector</td>
			<td>MEDNET</td>
			<td>NVFMLLPC</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Imaging software Version 5.02.00</td>
		</tr>
		<tr>
			<td>NOA81</td>
			<td>Thorlabs</td>
			<td>NOA81</td>
			<td>Fast Curing Optical Adhesive for tube system assembly</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
			<td></td>
		</tr>
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			<td>PCO edge 4.2 M-USB-HQ-PCO</td>
			<td>pco</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td>in-house prepared</td>
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			<td>Plasma Cleaner</td>
			<td>Diener Femto</td>
			<td>Pico-BRS</td>
			<td></td>
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		<tr>
			<td>PLL(20 kDa)-g[3.5]-PEG(2 kDa)</td>
			<td>SuSoS AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>silicon wafer mit mircorstructures</td>
			<td></td>
			<td></td>
			<td>in-house fabricated</td>
		</tr>
		<tr>
			<td>Sola Light Engine</td>
			<td>Lumencor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sticky slide VI 0.4&#160;</td>
			<td>ibidi</td>
			<td>80608</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>1673921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tango 2</td>
			<td>M&#228;rzh&#228;user</td>
			<td>00-24-626-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td></td>
			<td></td>
			<td>in-house prepared</td>
		</tr>
		<tr>
			<td>uncoated coverslips</td>
			<td>ibidi</td>
			<td>10813</td>
			<td></td>
		</tr>
		<tr>
			<td>Injekt-F Solo, 1 mL</td>
			<td>Omilab</td>
			<td>9166017V</td>
			<td>with replacement sporn</td>
		</tr>
	</tbody>
</table>

live-cell imaging of single-cell arrays (lisca) - a versatile technique to quantify cellular kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

The LISCA approach enables to efficiently collect fluorescence time courses from single cells. As a representative example we outline how the LISCA method is applied to measure single-cell eGFP expression after transfection. The data of the LISCA experiment is used to assess mRNA delivery kinetics, which is important for the development of efficient mRNA drugs.
In particular we demonstrate the different impact of two lipid-based mRNA delivery systems with respect to the time point of translati...

Watch this Scientific Journal Video about Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics at JoVE.com

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

Live-Cell Imaging of Single-Cell Arrays called LISCA are also an automated readout of the fluorescent time courses of hundreds of individual cells. The advantage of the approach is that individual cell responses are recorded from spatially isolated cells in identical micro environments, allowing an efficient emission analysis with great statistics. To fabricate a single-cell array, use a scalpel to cut a single PDMs masterpiece containing six micro powder stripes out of the PDMs layer, and place the masterpiece on the lab bench with the micro pattern facing up.Use a razor blade to cut each of the six micropattern stripes into a PDMs stamp, cutting off some of the patterned areas to ensure that the edges of the stamp are open. Next, carefully scratch the protective foil of the cover slip of a six chamber slide to mark the channel positions of the slide and place the cover slip on the bench with the protective foil facing down. Use tweezers to place the stamps on the cover slip at the marked channel positions, with the micro patterns facing down, and check the attachment of the stamps under a microscope.The squares in contact with the slide will appear darker than the interspace. The pattern quality is decreased by squares that are not properly attached to the slide. If the stamps have securely attached treat the cover slip and the stamps with oxygen plasma at 0.2 millibars of pressure and approximately 40 Watts for three minutes to make the surfaces between the PDMs stamps and the cover slip hydrophilic.At the end of the plasma cleaning, place the cover slip into a biosafety cabinet and add 15 microliters of pegylated poly-l-lysine solution to each stamp so that the solution is absorbed into the hydrophilic pattern of the stamp. After 20 minutes, rinse the stamps with one milliliter of ultrapure water. Use tweezers to remove the stamps from the slide and rinse the cover slip a second time with a second milliliter of ultrapure water.When the cover slip has dried completely, attach a six channel sticky slide to the cover slip taking care that the micro patterned areas align with the bottom of the channels. And add 40 microliters of PBS and 40 microliters of fibronectin solution to each channel. To mix the two solutions, remove 40 microliters from one reservoir and add it to the opposite reservoir of the same channel three times to generate a homogenous solution.When all of the channels have been mixed, incubate the slide for 45 minutes at room temperature before washing each channel three times with 120 microliters of PBS per wash. Exchange the PBS to 37 degrees Celsius, fully supplemented cell growth medium in the channels by adding 120 microliters medium to one reservoir and remove it from the opposite reservoir. Add 40 microliters of a 400, 000 cells per milliliter cells suspension of interest and 40 microliters of cell growth medium to each channel, and mix the cell solution with the medium as demonstrated.After mixing, remove 40 microliters of suspension from each channel so that only the channels are filled with cell suspension, and place the slide into a cell culture incubator. After one hour, check for cell adhesion by phase contrast microscopy, and add 120 microliters of 37 degrees Celsius cell growth medium to each channel, then return the slide to the cell culture incubator for three more hours. To set up a perfusion system, connect a one milliliter syringe filled with 37 degrees Celsius cell growth medium to the inlet tube of the system, and use the valve to fill the tube with medium.Connect the inlet tube to the reservoir of one channel, taking care that no air bubbles are trapped, and connect a serial connector to the reservoir opposite to the inlet tube of the current channel. Connect the rest of the channels as demonstrated until the required number of channels are connected in series. And connect the outlet tube directly to the free reservoir of the final channel.Then fill the connected tube with medium to confirm that the perfusion system does not leak. For time-lapse imaging of the cells, to set up a time lapse protocol for recording a phase contrast image and a fluorescence image, set the optical configuration for phase contrast imaging and fluorescence imaging. Use a 10 times objective, the appropriate fluorescence filters and an automated focus correction system to ensure a better image quality for the longterm measurements.Select a 10 millisecond exposure time for the phase contrast imaging and a 750 millisecond exposure time for recording the EGFP fluorescence intensity. Set a time schedule with a 10-minute time interval between the consecutive loops through the position list and an observation time of 30 hours. Next, place the six channels slide on the single cellar raise in the sample holder of the 37 degrees Celsius heating chamber of the microscope and tape the perfusion system tubing to the stage.Insert the free ends of the outlet tubes into a 15 milliliter conical tube to collect the liquid waste. And set the position list for the scanning time lapse measurement, taking care that the number of positions will be able to be scanned within the defined time interval between consecutive loops through the position list. Then start the time lapse measurement.For transfection of the cells with a fluorescent marker, use a syringe to flush the tubing system with one milliliter of 37 degrees Celsius PBS, taking care that the microscope stage doesn't move. After flushing, fill the system with 300 microliters of serum reduced medium supplemented with the mRNA of interest, to a final concentration of 0.5 nanograms of mRNA per microliter, and allow the lipoplexes to incubate for one hour. At the end of the incubation, flush the system with one milliliter of 37 degrees Celsius complete cell growth medium to remove any unbound mRNA.At the end of the analysis, view the channels listed in the channel menu and scroll through the timeframes to inspect that preselected cells and their integrated fluorescent signals. Click on a cell of interest to highlight its fluorescence time course, or click on a time course to find the corresponding cell. Press shift while clicking on a cell to select or de-select it.De-select any cells that are not viable or not confined to an adhesion spot or that are attached to another cell to exclude them from further analysis. When all of the cells are selected or deselected appropriately, save the single cell time courses for the cell area and the integrated fluorescence in an appropriate directory. To quantify the translation onset time after the transfection, and the strength of the cellular EGFP expression, a three-stage translation model can be employed.The model solution for EGFP can be fitted to single cell time courses as illustrated. Here, histograms of the translation onset time and the expression rate of lipoplex transfected cells can be observed. As both parameters are estimated for each cell, the correlation of these parameters can be analyzed as demonstrated in the scatter plot, and compared to cells transfected with lipid nanoparticles.As illustrated, cells transfected with lipid nanoparticles exhibit less cell-to-cell variability compared to cells transfected with lipoplexes. And the population average demonstrates a faster onset of translation, as well as a higher expression rate. Our approach can also be adapted to alternative micro-patterning methods.Most important for LISCA is a good cell confinement and a combination of automated image analysis with a convenient user supervision. As you have seen, cell behavior is heterogeneous. And single-cell time lapse movies provide access to unbiased dynamics of the inherent biochemical networks.For example, in green expression, apoptosis or cell killing assays.

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Research

JoVE Journal

Biology

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...