LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

Dakin, Kenneth; Zhao, YuRui; Li, Wen Hong

doi:10.1038/nmeth730

Cited by 88 publications

(88 citation statements)

References 29 publications

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“…At the early time points (0-20 min), the transfer rates are slower than the later stages. The trend of transfer rate is different from what has been previously reported from local activation of molecular fluorescent probe experiments, which showed consistent dye transfer constant over the time period (23). The reason lies in that the acoustic well experiments enable the examination of dye transfer immediately after cell-cell contacts.…”

Section: Resultscontrasting

confidence: 97%

Controlling cell–cell interactions using surface acoustic waves

Guo

French

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

353

257

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The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.cell-cell interaction | intercellular communication | surface acoustic waves | acoustic tweezers | acoustofluidics M ulticellular systems rely on the interaction between cells to coordinate cell signaling and regulate cell functions. Understanding the mechanism and process of cell-cell interaction is critical to many physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes (1-3). Despite significant advances in this field, to further understand how cells interact and communicate with each other, a robust, biocompatible method to precisely control the spatial and temporal association of cells and to create defined cellular assemblies is urgently needed (4). Although several methods have been used to pattern cells, limitations still exist for the demonstrated methods including those that make use of optical, electrical, magnetic, hydrodynamic, and contact printing technologies (5-9). Firstly, most of the methods require modification of the cell's native state. The magnetic assembly method, for example, requires cells to be labeled with magnetic probes. Dielectrophoresis typically requires the use of a special medium (e.g., nonconductive) which may lack essential nutrients or have biophysical properties (such as the osmolality) that may adversely affect cell growth or physiology (6). Optical tweezers provide a label-free and contactless approach, but typically require high laser power to manipulate cells, leading to a high risk of cell damage (5). Secondly, the working principles of the existing technologies mostly preclude the combination of high precision and high throughput into a single ...

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Section: Resultscontrasting

confidence: 97%

Controlling cell–cell interactions using surface acoustic waves

Guo

French

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

353

257

View full text Add to dashboard Cite

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“…Different strategies have been used to evaluate gap junction functionality, including metabolic cooperation, radioactive nucleotide transfer, fluorescent dye microinjection, scrape-loading, dual whole cell patch-clamp electrophysi-ology, standard eletrophysiology, fluorescence recovery after photobleaching, local activation of a molecular fluorescent probe, and the so-called ''parachute assay,'' in which cells labeled with a gap junction permeant indicator are plated atop unlabeled cells (9)(10)(11)(12)(13)(14).…”

Section: Resultsmentioning

confidence: 99%

Flow cytometry analysis of gap junction‐mediated cell–cell communication: Advantages and pitfalls

et al. 2006

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Background: Since the first morphological description of the gap junctions use electron microscopy, a considerable number of techniques has been introduced to evaluate gap junction channel functionality, many of which use dye transfer techniques, such as dye injection and fluorescent dye transfer, analyzed by flow cytometry. Methods: To analyze dye transfer, generally one population of cells is incubated with calcein-AM (0.5 lM) for 30 min at 37°C, and the other population was incubated with the lipophilic dye DiIC 18 (3) (10 lM) for 1 h at 37°C; after incubation, these cells were washed five times with PBS and cocultured for different times, and then the dye transfer was analyzed by flow cytometry.

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“…In this experiment, we formed heterotypic cell pairs with controlled morphology to study material transport through gap junction intercellular communication (GJIC) (30). Studying GJIC at the single-cell level is significant and challenging and has traditionally been carried out using techniques such as microinjection (31), dual whole-cell patch clamp (32), gap-fluorescence recovery after photobleaching (33), and local activation of a molecular fluorescent probe (34). However, such studies are impeded by the uncontrollability of gap junction formation and conflicting aims of reducing invasiveness while maintaining high throughput.…”

Section: Gap Junction Intercellular Communication In Cell Pairs With mentioning

confidence: 99%

Block-Cell-Printing for live single-cell printing

Zhang¹,

Chou

Xia

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

127

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A unique live-cell printing technique, termed "Block-Cell-Printing" (BloC-Printing), allows for convenient, precise, multiplexed, and high-throughput printing of functional single-cell arrays. Adapted from woodblock printing techniques, the approach employs microfluidic arrays of hook-shaped traps to hold cells at designated positions and directly transfer the anchored cells onto various substrates. BloC-Printing has a minimum turnaround time of 0.5 h, a maximum resolution of 5 μm, close to 100% cell viability, the ability to handle multiple cell types, and efficiently construct protrusion-connected single-cell arrays. The approach enables the large-scale formation of heterotypic cell pairs with controlled morphology and allows for material transport through gap junction intercellular communication. When six types of breast cancer cells are allowed to extend membrane protrusions in the BloC-Printing device for 3 h, multiple biophysical characteristics of cells-including the protrusion percentage, extension rate, and cell length-are easily quantified and found to correlate well with their migration levels. In light of this discovery, BloC-Printing may serve as a rapid and high-throughput cell protrusion characterization tool to measure the invasion and migration capability of cancer cells. Furthermore, primary neurons are also compatible with BloC-Printing.cell array | cell communication | protrusion profiling | neuron patterning C urrent high-throughput screening of cell function and heterogeneity and in vitro cell-cell communication studies requires routine generation of large-scale single-cell arrays with high precision and efficiency, single-cell resolution, multiple cell types, and maintenance of cell viability and function (1, 2). Several approaches have been designed for this purpose, e.g., inkjet cell printing (3-6), surface engineering (7-15), and physical constraints (16-23). However, finding a method that completely satisfies the above requirements remains a challenge. Potentially useful and convenient tools may be available by adapting traditional printing tools to cell printing. In particular, woodblock printing is an efficient and convenient technology that revolutionized the printing world more than 1,800 y ago and was extended to microcontact molecular printing ∼20 y ago (24). However, application of the block-printing concept to cells has never been achieved. The main challenges are (i) inking cells to their molds with precision and maintaining viability, (ii) evenly and gently applying and transferring the cells to a substrate and successfully lifting off the mold without detaching the cells, and (iii) maintaining cell functions after printing.We report here the development and testing of a technology called "Block-Cell-Printing" (BloC-Printing), which involves directly inking cells to a predesigned mold and then transferring the cells to a substrate. We overcome the challenges described above by flow patterning, instead of pressing, the ink objects, as in woodblock or microcontact printing. By ...

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LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

Cited by 88 publications

References 29 publications

Controlling cell–cell interactions using surface acoustic waves

Controlling cell–cell interactions using surface acoustic waves

Flow cytometry analysis of gap junction‐mediated cell–cell communication: Advantages and pitfalls

Block-Cell-Printing for live single-cell printing

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