eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Microfluidic device preparation.
<ol>
	<li>Microfluidic Cell Migration Assay Preparation
	<ol>
		<li>Prepare 50 &#181;g/mL fibronectin solution by diluting 50 &#181;L of stock fibronectin solution (1 mg/mL) to 950 &#181;L Dulbecco&#39;s phosphate-buffered saline (DPBS) inside a biosafety cabinet.</li>
		<li>Prepare the mig...

a microfluidic chip-based method for rapid neutrophil chemotaxis analysis using whole blood

Source: Yang, K., et al. <a href="https://app.jove.com/v/55615">An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood.</a> J. Vis. Exp. (124), (2017)
This video demonstrates neutrophil chemotaxis assay using an all-on-chip method. The method allows direct isolation of neutrophils from the whole blood by immunomagnetic negative selection followed by chemotaxis assay on a microfluidic chip.

<img alt="Figure 1" src="/files/ftp_upload/21903/21903_Figure1.jpg" /> 
Figure 1: Illustration of the all-on-chip method for neutrophil chemotaxis analysis. (A) Illustration of the microfluidic device. The device includes two layers. The first layer (4 &#181;m high) defines the cell docking barrier channel to trap the cells beside the gradient channel. The second layer (60 &#181;m high) defines the gradient generating channel, the port and channel fo...

A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Microfluidic device preparation.

...

To a whole blood sample, add specific antibody complexes directed against blood cells except neutrophils.
Add magnetic particles that bind to the antibody complex-bound blood cells.
Transfer the immunomagnetically tagged sample to the cell-loading port of a microfluidic chip.
Attach magnetic disks to the cell-loading port.
The magnetic field attracts the immunomagnetically-tagged cells, trapping them to the port&#39;s side walls.
The neutrophils, being untagged, flow into the chip and become trapped at the cell-docking area.
Add fluorescently-labeled chemoattractant solution and migration medium to designated reservoirs. The liquids flow through the microfluidic channels, forming a concentration gradient.
When the chemoattractant binds to the neutrophil receptor, it triggers intracellular changes, causing neutrophils to reorganize their cytoskeleton and extend pseudopods.
These pseudopods act as feet, propelling the neutrophils toward higher chemoattractant concentrations &#8212; a process called chemotaxis.
Use appropriate microscopy techniques to monitor neutrophil migration in response to the chemoattractant gradient.

a-microfluidic-chip-based-method-for-rapid-neutrophil-chemotaxis

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

JoVE Journal

Bioengineering

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood

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