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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 migration medium by mixing 9 mL of Roswell Park Memorial Institute medium (RPMI-1640) and 1 mL of RPMI-1640 with 4% bovine serum albumin (BSA).</li>
		<li>Remove the deionized water from the device.</li>
		<li>Add 100 &#181;L fibronectin solution to the device from the outlet. Wait 3 min to ensure that all the channels are filled with fibronectin solution. Place the microfluidic device in a covered Petri dish for 1 h at room temperature.</li>
		<li>Remove the fibronectin solution from the device. Add 100 &#181;L migration medium from the outlet. Wait 3 min to ensure that all the channels are filled with migration medium.</li>
		<li>Incubate the device for another 1 h at room temperature; the device is then ready for the chemotaxis experiment.</li>
	</ol>
	</li>
	<li>Chemoattractant solution preparation for chemotaxis experiment.&#8203;
	<ol>
		<li>Prepare 100 nM fMLP solution in a total of 1 mL migration medium. Mix 5 &#181;L of stock FITC-Dextran (10 kDa, 1 mM) with the fMLP solution in a 1.5 mL tube. 
		NOTE: FITC-Dextran is used for gradient measurement. Alternatively, use Rhodamine as the gradient indicator. The fMLP chemoattractant solution is then ready for the chemotaxis experiment.</li>
	</ol>
	</li>
	<li>Sputum sample preparation. 
	NOTE: Neutrophil chemotaxis induced by a gradient of sputum samples from COPD patients was tested as a clinical diagnostic application of this all-on-chip method.
	<ol>
		<li>Obtain a human ethics protocol to collect sputum samples from COPD patients. 
		NOTE: We obtained approvals to collect samples at the Seven Oaks General Hospital in Winnipeg (approved by the University of Manitoba).</li>
		<li>Obtain the informed written consent forms from all subjects.</li>
		<li>Collect COPD patients&#39; spontaneous sputum samples. Place 500 &#181;L sputum sample in a 1.5 mL tube.</li>
		<li>Add 500 &#181;L 0.1% dithiothreitol in the 1.5 mL tube and gently mix. Place the tube in a water bath at 37 &#176;C for 15 min.</li>
		<li>Centrifuge the sample at 753 x g for 10 min and then collect the supernatant. Centrifuge the supernatant at 865 x g for 5 min and then collect the final supernatant. Store the collected supernatant inside a -80 &#176;C freezer before use.</li>
		<li>When ready for the chemotaxis experiment, thaw the sputum solution; transfer 900 &#181;L migration medium to a 1.5 mL tube and mix with 100 &#181;L sputum solution inside a biosafety cabinet; the sputum solution is then ready for the chemotaxis experiment.</li>
	</ol>
	</li>
	<li>Blood sample collection.
	<ol>
		<li>Obtain a human ethics protocol to collect blood samples from healthy donors. Obtain the informed written consent forms from all blood donors. 
		NOTE: Here samples were obtained at the Victoria General Hospital in Winnipeg (approved by the Joint-Faculty Research Ethics Board at the University of Manitoba).</li>
		<li>Collect the blood sample by venipuncture and put the sample into an EDTA-coated tube. Keep the tube in a biosafety cabinet before the experiment.</li>
	</ol>
	</li>
</ol>
2. All-on-chip Chemotaxis Assay Operation
<ol>
	<li>On-chip cell isolation (Figure 1B).</li>
	<li>Place 10 &#181;L whole blood in a 1.5 mL tube inside a biosafety cabinet. 
	NOTE: Details of blood sample collection are in section 1.4.</li>
	<li>Add 2 &#181;L antibody cocktail (Ab) and 2 &#181;L magnetic particles (MP) from the neutrophil isolation kit (see the table of materials) into the 1.5 mL tube and gently mix; this will label cells in the blood except for the neutrophils.</li>
	<li>Incubate the blood-Ab-MP mixture for 5 minutes at room temperature. 
	NOTE: This will magnetically tag the antibody-labeled cells in the blood.</li>
	<li>Attach two small magnetic disks to the two sides of the cell loading port of the device. Aspirate the medium from all ports of the device.</li>
	<li>Slowly pipette 2 &#181;L blood-Ab-MP mixture into the microfluidic device from the cell loading port. 
	NOTE: The magnetically labeled cells are trapped to the side walls of the cell loading port while neutrophils will flow into the device and become trapped at the cell docking structure.</li>
	<li>Wait a few minutes until enough neutrophils are trapped in the cell docking area.</li>
</ol>
3. Chemotaxis assay (Figure 1C).
<ol>
	<li>Place the microfluidic device on the temperature-controlled microscope stage at 37 &#176;C.</li>
	<li>Add 100 &#181;L chemoattractant solution (fMLP or sputum solution) and 100 &#181;L migration medium to their designated inlet reservoirs using two pipettors; this will generate a chemoattractant gradient in the gradient channel by continuous laminar flow-based chemical mixing assisted by a pressure balancing structure. 
	NOTE: Details of the sputum collection from COPD patients are in section 1.3.
	<ol>
		<li>For the medium control experiment, only add migration medium to both of the inlet reservoirs.</li>
	</ol>
	</li>
	<li>Acquire the fluorescence image of FITC-Dextran in the gradient channel.</li>
	<li>Import the image to ImageJ software using the command &#34;File|Open&#34;.</li>
	<li>Measure the fluorescence intensity profile across the gradient channel using the command &#34;Analyze|Plot Profile&#34;.</li>
	<li>Export the measurement data to a spreadsheet for further plotting.</li>
	<li>Incubate the device on the temperature-controlled microscope stage or in a conventional cell culture incubator for 15 min.</li>
	<li>Image the gradient channel using a 10X objective to record the cells&#39; final positions for data analysis.</li>
	<li>If needed, record the cell migration in the device by time-lapse microscopy.</li>
</ol>
4. Cell Migration Data Analysis (Figure 1C)
<ol>
	<li>Analyze the chemotaxis assay by calculating the cell migration distance from the docking structure as described below. See Figure 1C.</li>
	<li>Import the image into NIH ImageJ software (ver. 1.45).</li>
	<li>Select the center of each cell that moved into the gradient channel.</li>
	<li>Measure the coordinates of the selected cells for their final positions. Measure the coordinate of a point at the edge of the docking structure as the initial reference position.</li>
	<li>Export the measured coordinate data to spreadsheet software (e.g. Excel). Calculate the migration distance of the cells as the difference between a cell&#39;s final position and the initial reference position along the gradient direction.</li>
	<li>Calibrate the distance to a micrometer. Calculate the average and deviation of the migration distance of all cells as a measure of chemotaxis.</li>
	<li>Compare the migration distance in the presence of a chemoattractant gradient to the medium control experiment using the Student&#39;s t-test.</li>
	<li>If the time-lapse images of the cell migration are recorded, the cell migration and chemotaxis can be further analyzed by cell tracking analysis. 
	NOTE: The materials required to construct and perform the all-on-chip chemotaxis assay are detailed in the table of materials.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Device fabrication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mask aligner</td>
			<td>ABM</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner</td>
			<td>Solitec</td>
			<td>5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>VWR</td>
			<td>11301-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dessicator</td>
			<td>Fisher Scientific</td>
			<td>08-594-15A</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2000 thinner</td>
			<td>Microchem</td>
			<td>SU-8 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2025 photoresist</td>
			<td>Microchem</td>
			<td>SU-8 2025</td>
			<td></td>
		</tr>
		<tr>
			<td>(tridecafluoro-1,1,2,2- tetrahydrooctyl) trichlorosilane</td>
			<td>Gelest</td>
			<td>78560-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Silicon, Inc</td>
			<td>LG2065</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting pad</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made</td>
		</tr>
		<tr>
			<td>Punchers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>On-chip cell isolation and chemotaxis assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Fisher Scientific</td>
			<td>SH3025502</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Fisher Scientific</td>
			<td>SH3002802</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>SH3057402</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>VWR</td>
			<td>CACB356008</td>
			<td></td>
		</tr>
		<tr>
			<td>fMLP</td>
			<td>Sigma-Aldrich</td>
			<td>F3506-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic disks</td>
			<td>Indigo Instruments</td>
			<td>44202-1</td>
			<td>5 mm in diameter, 1 mm thick</td>
		</tr>
		<tr>
			<td>FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD10S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine</td>
			<td>Sigma-Aldrich</td>
			<td>R4127-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa stain solution</td>
			<td>Rowley Biochemical Inc</td>
			<td>G-472-1-8OZ</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Direct Human Neutrophil Isolation Kit</td>
			<td>STEMCELL Technologies Inc</td>
			<td>19666</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti-U inverted fluorescent microscope</td>
			<td>Nikon</td>
			<td>Ti-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope environmental chamber.</td>
			<td>InVivo Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Nikon</td>
			<td>DS-Fi1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Source: Yang, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.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

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

74 Views. Fonte: Yang, K., et al. Un metodo all-on-chip per l'analisi rapida della chemiotassi dei neutrofili direttamente da una goccia di sangue. J. Vis. Exp. (124), (2017) Questo video mostra il test della chemiotassi dei neutrofili utilizzando un metodo all-on-chip. Il metodo consente l'isolamento diretto dei neutrofili dall'intero sangue mediante selezione immunomagnetica negativa seguita da un test di chemiotassi su un chip microfluidico. 

Video: Un metodo basato su chip microfluidico per l'analisi rapida della chemiotassi dei neutrofili utilizzando sangue intero - Concept

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.

Microscopia di trazione integrata con microfluidica per la migrazione collettiva chemiotattica

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

Ricerca

JoVE Journal

Bioingegneria

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.

Microarray di bioparticelle per l'analisi chemiotattica e molecolare dello sciame neutrofilo umano in vitro

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.

Imaging G protein-coupled recettore-mediata chemiotassi ed i suoi eventi di segnalazione nelle cellule HL60 neutrofili-like

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

Trascrizione

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