This work is in part supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). We thank the Clinical Institute of Applied Research and Education at the Victoria General Hospital in Winnipeg and Seven Oaks General Hospital in Winnipeg for managing clinical samples from human subjects. We thank Dr. Hagit Peretz-Soroka for helpful discussion about the assay operation strategies.
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We thank Professor Carolyn Ren and Dr. Xiaoming (Cody) Chen from the University of Waterloo for their generous support in the filming process.

All human sample collection protocols were approved by the Joint-Faculty Research Ethics Board at the University of Manitoba, Winnipeg.
1. Microfluidic Device Fabrication (Figure 1A)
<ol>
	<li>Design and print transparency mask.
	<ol>
		<li>Design the device as detailed previously25. See Figure 1A. 
		NOTE: 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 for cell loading, the chemical inlet reservoirs and waste outlet. The alignment marks are designed for the two layers. For the second layer, the length and width of the upstream serpentine input channel is 60 mm and 200 &#181;m, respectively; the length and width of the downstream serpentine input channel is 6 mm and 280 &#181;m, respectively.</li>
		<li>Print the first and second layer features to a transparency mask using a high-resolution printer. 
		NOTE: The printing resolution depends on the minimum features in the design. In the current design, 32,000 dpi was chosen for 10 &#181;m minimum feature.</li>
	</ol>
	</li>
	<li>Clean the silicon wafer.
	<ol>
		<li>Place a 3 in silicon wafer into the plasma cleaner. Apply vacuum for 3 min.</li>
		<li>Turn on the plasma power and set the level to HIGH. Use the oxygen plasma to treat the silicon wafer for 30 min.</li>
		<li>Turn off the plasma cleaner and take out the silicon wafer; the silicon wafer is ready for fabricating the device mold.</li>
	</ol>
	</li>
	<li>Fabricate the first layer by photolithography in a cleanroom facility. 
	NOTE: The exact fabrication parameters may vary depending on the fabrication facility.
	<ol>
		<li>Dilute 10 mL SU-8 2025 with 10 mL SU-8 2000 in a glass breaker to prepare the designed photoresist. Leave the mixture in the fume hood for 10 min until the bubbles disappear.</li>
		<li>Carefully place the cleaned silicon wafer on the spinner with the suitable chuck and apply the vacuum to immobilize the silicon wafer.</li>
		<li>Carefully pour 3 mL of the photoresist mixture onto the center of the silicon wafer. Spin at 500 rpm for 5 s. Then spin at 3,000 rpm for 30 s to obtain a final 4 &#181;m thickness photoresist coating on the silicon wafer.</li>
		<li>Carefully remove the silicon wafer from the spinner and bake the silicon wafer on a hotplate for 4 min at 95 &#176;C.</li>
		<li>Carefully place the silicon wafer on a mask aligner and set the UV exposure time to 6 s. Carefully attach the transparency mask of the first layer on a transparent glass plate using adhesive tape.</li>
		<li>Gently place the transparent glass plate with the attached mask to the aligner and align the mask with the silicon wafer. Expose the silicon wafer to the UV to pattern the cell docking structure.</li>
		<li>Carefully remove the glass plate and take out the exposed silicon wafer. Bake the silicon wafer on a hotplate for 4 min at 95 &#176;C.</li>
		<li>Transfer the silicon wafer to a fume hood and place it in a glass pan containing the SU-8 developer. Gently shake this for 30 s.</li>
		<li>Clean the silicon wafer using fresh SU-8 developer followed by isopropyl alcohol (IPA) inside the fume hood. Dry the silicon wafer by nitrogen gas inside the fume hood; the first layer is ready.</li>
	</ol>
	</li>
	<li>Fabricate the second layer on the first layer.
	<ol>
		<li>Use adhesive tape to cover the alignment marks on the first layer. Carefully place the silicon wafer with the first layer on the vacuum chuck of the spinner and apply vacuum to immobilize the silicon wafer.</li>
		<li>Pour 3 mL of SU-8 2025 photoresist on the silicon wafer. Spin the silicon wafer at 500 rpm for 5 s. Then spin at 2000 rpm for 30 s to obtain a final 60 &#181;m thickness photoresist coating on the silicon wafer.</li>
		<li>Carefully remove the silicon wafer from the spinner and transfer it to a hotplate; bake at 65 &#176;C for 2 min.</li>
		<li>Gently remove the adhesive tape to expose the alignment marks on the first layer. Place the silicon wafer on a hotplate and bake at 95 &#176;C for 6 min.</li>
		<li>Carefully place the silicon wafer on a mask aligner and set the UV exposure time to be 18 s.</li>
		<li>Carefully attach the transparency mask of the second layer on a transparent glass plate using adhesive tape.</li>
		<li>Carefully place the glass plate with the attached mask to the aligner, and align the mask and the first layer on the silicon wafer by the crossing alignment marks using the inspection microscope of the aligner.</li>
		<li>Expose the photoresist coated silicon wafer to UV to pattern the cell loading and gradient channels.</li>
		<li>Carefully remove the glass plate and take out the silicon wafer. Bake the silicon wafer on a hotplate at 65 &#176;C for 2 min and then transfer the silicon wafer to another 95 &#176;C hot plate and bake for 6 min.</li>
		<li>Transfer the silicon wafer to the fume hood and place it in a glass pan containing the SU-8 developer. Gently shake this for 6 min.</li>
		<li>Clean the silicon wafer using fresh SU-8 developer followed by IPA inside the fume hood.</li>
		<li>Dry the silicon wafer using nitrogen gas inside the fume hood. Place the silicon wafer on a hotplate and hard bake the mold at 150 &#176;C for 30 min; the second layer is ready.</li>
	</ol>
	</li>
	<li>Master mold surface modification. 
	NOTE: A silanization surface modification step is applied to the SU-8 mold to facilitate polydimethylsiloxane (PDMS) release from the mold in soft-lithography.
	<ol>
		<li>Take 10 &#181;L of tridecafluoro-1,1,2,2-tetrahydrooctyl (trichlorosilane) solution in a micropipette tip. Put the micropipette tip into a 15 mL plastic tube and loosen the cap of the tube.</li>
		<li>Place the tube and the SU-8 patterned silicon wafer inside a desiccator and apply the vacuum for 1 h; the mask mold is ready for fabricating the PDMS device.</li>
	</ol>
	</li>
	<li>Fabricate the PDMS device.
	<ol>
		<li>Prepare the PDMS solution by mixing 40 g PDMS base and 4 g curing agent in a plastic beaker. Place the prepared SU-8 master mold in a Petri dish and carefully pour 44 g PDMS solution onto the mold.</li>
		<li>Place the Petri dish in a desiccator and apply vacuum to degas the PDMS solution for 20 min. Then place the Petri dish in an oven and cure the PDMS at 80 &#176;C for 2 h.</li>
		<li>After baking, take out the Petri dish and place it on a clean bench. Carefully cut and peel off the PDMS slab from the SU-8 mold.</li>
		<li>Punch out the cell loading port using a 3 mm diameter puncher. Punch out the chemical inlet reservoirs and the waste outlet using a 6 mm diameter puncher.</li>
		<li>Remove the dust on the surface of the PDMS slab using adhesive tape. Place the PDMS slab and a clean glass slide into the plasma machine. Apply the vacuum for 3 min.</li>
		<li>Turn on the plasma power and set the level to HIGH. Gently adjust the air valve and plasmatreat the PDMS slab and the glass slide for 3 min.</li>
		<li>Turn off the plasma power and release the vacuum. Carefully take out the PDMS slab and the glass slide using tweezers.</li>
		<li>Immediately place the PDMS slab (with channel structures face-down) on top of the glass slide; gently press the PDMS slab to bond it to the glass. Fill the microfluidic channel with deionized water immediately; the microfluidic device fabrication and assembly are completed.</li>
	</ol>
	</li>
</ol>
2. Microfluidic Cell Migration Assay Preparation
<ol>
	<li>Microfluidic device 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's phosphate-buffered saline (DPBS) inside a biosafety cabinet.</li>
		<li>Prepare the migration medium by mixing 9 mL 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.
	<ol>
		<li>Prepare 100 nM fMLP solution in total 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 chemotaxis experiment.</li>
	</ol>
	</li>
	<li>Sputum sample preparation. 
	NOTE: Neutrophil chemotaxis induced by a gradient of sputum sample 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' 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 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 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>
3. All-on-chip Chemotaxis Assay Operation
<ol>
	<li>On-chip cell isolation (Figure 1B).
	<ol>
		<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 2.4.</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 the neutrophils.</li>
		<li>Incubate the blood-Ab-MP mixture for 5 min at room temperature. 
		NOTE: This will magnetically tag the antibody labeled cells in 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 at the cell docking area.</li>
	</ol>
	</li>
	<li>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 2.3.
		<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 "File|Open".</li>
		<li>Measure the fluorescence intensity profile across the gradient channel using the command "Analyze|Plot Profile".</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' final positions for data analysis.</li>
		<li>If needed, record the cell migration in the device by time-lapse microscopy.</li>
	</ol>
	</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 a spreadsheet software (e.g. Excel). Calculate the migration distance of the cells as the difference between a cell'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'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 analysis15. 
	NOTE: The materials required to construct and perform the all-on-chip chemotaxis assay are detailed in the table of materials.</li>
</ol>

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There are no conflicts of interest to disclose.

In this paper, a detailed protocol to directly isolate neutrophils from whole blood followed by the chemotaxis test, all on a single microfluidic chip, was described. This method offers useful features in its easy operation, negative selection of high purity neutrophils, rapid sample-to-result chemotaxis test, reduced reagents and sample consumption, and accurate cell migration data analysis. As a rough estimate, at least 25% of the neutrophils from the input whole blood sample effectively entered the docking structure i...

Chemotaxis, a process of directed cell migration to soluble chemical concentration gradient, is critically involved in many biological processes including immune response1,2,3, tissue development4 and cancer metastasis5. Neutrophils are the most abundant white blood cell subset and play crucial roles in enabling the body&#39;s innate host defense functions, as well as in mediating adaptive immune responses6,7. Neutrophils are equipped with highly-regulated chemotactic machinery allowing these motile immune cells to respond to both pathogen-derived chemoattractants (e.g. fMLP) and host-derived chemoattractants (e.g. interleukin-8) throughchemotaxis8. Neutrophil migration and chemotaxis mediate various physiological problems and diseases such as inflammation and cancers1,9. Thus, the accurate assessment of neutrophil chemotaxis provides an important functional readout for studying the neutrophil biology and the associated diseases.
Compared to the widely-used conventional chemotaxis assays (e.g. transwell assay10), the microfluidic devices show great promise for quantitative evaluation of cell migration and chemotaxis owing to the precisely controlled chemical gradient generation and miniaturization11,12,13. Over the last two decades or so, various microfluidic devices have been developed to study the chemotaxis of different biological cell types, especially neutrophils11. Significant effort was devoted to characterizing neutrophil migration in spatiotemporally complex chemical gradients that were configured in the microfluidic devices14,15. Interesting strategies were also developed to study directional decision making by neutrophils using the microfluidic devices16.Aside from biologically-oriented research, the applications of microfluidic devices have been extended to test clinical samples for disease evaluation17,18,19. However, the use of many microfluidic devices is limited to specialized research laboratories and requires lengthy neutrophil isolation from large volume of blood samples. Therefore, there has been a growing trend of developing integrated microfluidic devices for rapid neutrophil chemotaxis analysis directly from a drop of whole blood20,21,22,23,24.
Toward this direction, an all-on-chip method was developed that integrates the magnetic negative neutrophil purification and the subsequent chemotaxis assay on a single microfluidic device25. This all-on-chip method has the following novel features: 1) in contrast to previous on-chip strategies that isolate neutrophils from the blood by adhesion-based cell capture or cell size-based filtering20,22, this new method permits high purity, on-chip magnetic separation of the neutrophils from small volumes of whole blood, as well as chemotaxis measurement upon chemoattractant stimulation; 2) the cell docking structure helps align the initial positions of the neutrophils close to the chemical gradient channel and permits simple chemotaxis analysis without single cell tracking; 3) the integration of the neutrophil isolation and chemotaxis assay on a single microfluidic device permits rapid sample-to-result chemotaxis analysis in 25 min when there is no interruption between experimental steps.
This paper provides a detailed protocol for the construction, operation and data analysis method of this all-on-chip chemotaxis assay. The paper demonstrates the effective use of the developed method for performing neutrophil chemotaxis by testing a known recombinant chemoattractant and complex chemotactic samples from patients, followed by a discussion on troubleshooting strategies, limitations and future directions.

<table><tbody><tr><td>&lt;strong&gt;Device fabrication&lt;/strong&gt;</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>Digital scale</td><td>Ohaus</td><td>CS200</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>SU-8 developer</td><td>Microchem</td><td>SU-8 developer</td><td></td></tr><tr><td>Si wafer</td><td>Silicon, Inc</td><td>LG2065</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Fisher Scientific</td><td>A416-4</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>Polydimethylsiloxane&lt;br/&gt; (PDMS)</td><td>Ellsworth Adhesives</td><td>2065622</td><td></td></tr><tr><td>Petri Dish</td><td>Fisher Scientific</td><td>FB0875714</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>N/A</td><td>N/A</td><td>Custom-made</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Source&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;On-chip cell isolation and chemotaxis assay&lt;/strong&gt;</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&lt;br/&gt; (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,&lt;br/&gt; 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>&lt;br/&gt; 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&lt;br/&gt; Neutrophil Isolation&lt;br/&gt; Kit</td><td>STEMCELL&lt;br/&gt; 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>

an all-on-chip method for rapid neutrophil chemotaxis analysis directly from a drop of blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

Neutrophils are negatively selected from a drop of whole blood directly in the microfluidic device. The purity of the isolated neutrophils was verified by on-chip Giemsa staining and the results showed the typical ring-shaped and lobe-shaped nuclei of neutrophils (Figure 2A)25. This indicates an effective on-chip neutrophil isolation at high purity from a small volume of whole blood. Furthermore, the docking structure can effectively a...

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An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

an-all-on-chip-method-for-rapid-neutrophil-chemotaxis-analysis

Research

JoVE Journal

Immunology and Infection

17.0K Views.  Chinese Academy of Sciences.  Dit artikel geeft de gedetailleerde methode om een ​​snelle neutrofiele chemotaxis-analyse te verrichten door de integratie van de on-chip neutrofiele isolatie uit volledig bloed en de chemotaxis-test op een enkele microfluïdische chip. 

Video: An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Bioengineering

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

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 ...

Screening of β2 Integrin Activation Inhibitors in Human Neutrophils

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

Author Spotlight: Development of a Method for Identifying Small Molecular Antagonists of &#946;2 Integrin Activation 

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing ...

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 ...

Biology

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

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