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.
<style type="text/css">
</style>
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>

<ol>
	<li>Kolaczkowska, E., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+and+function+in+health+and+inflammation.">Neutrophil recruitment and function in health and inflammation.</a> Nat Rev Immunol. 13 (13), 159-175 (2013).</li><li>Luster, A. D., Alon, R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+migration+in+inflammation:+present+and+future+therapeutic+targets.">Immune cell migration in inflammation: present and future therapeutic targets.</a> Nat Immunol. 6 (12), 1182-1190 (2005).</li><li>Griffith, J. W., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cells+in+motion:+migrating+toward+improved+therapies.">Targeting cells in motion: migrating toward improved therapies.</a> Eur. J. Immunol. 43 (6), 1430-1435 (2013).</li><li>Laird, D. J., von Andrian, U. H., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+trafficking+in+tissue+development,+growth,+and+disease.">Stem cell trafficking in tissue development, growth, and disease.</a> Cell. 132 (4), 612-630 (2008).</li><li>Condeelis, J., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+cell+movement+in+tumours.">Intravital imaging of cell movement in tumours.</a> Nat Rev Cancer. 3 (12), 921-930 (2003).</li><li>Kruger, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils:+between+host+defence,+immune+modulation,+and+tissue+injury.">Neutrophils: between host defence, immune modulation, and tissue injury.</a> PLoS Pathog. 11 (3), e1004651 (2015).</li><li>M&#243;csai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+novel+functions+of+neutrophils+in+immunity,+inflammation,+and+beyond.">Diverse novel functions of neutrophils in immunity, inflammation, and beyond.</a> J Exp Med. 210 (7), 1283-1299 (2013).</li><li>Foxman, E. F., Campbell, J. J., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistep+navigation+and+the+combinatorial+control+of+leukocyte+chemotaxis.">Multistep navigation and the combinatorial control of leukocyte chemotaxis.</a> J Cell Biol. 139 (7), 1349-1360 (1997).</li><li>Tazzyman, S., Niaz, H., Murdoch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-mediated+tumour+angiogenesis:+subversion+of+immune+responses+to+promote+tumour+growth.">Neutrophil-mediated tumour angiogenesis: subversion of immune responses to promote tumour growth.</a> Semin Cancer Biol. 23 (3), 149-158 (2013).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J Exp Med. 115 (3), 453-466 (1962).</li><li>Wu, J., Wu, X., Lin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+microfluidics-based+chemotaxis+studies.">Recent developments in microfluidics-based chemotaxis studies.</a> Lab Chip. 13 (13), 2484-2499 (2013).</li><li>Sackmann, E. K., Fulton, A. L., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+present+and+future+role+of+microfluidics+in+biomedical+research.">The present and future role of microfluidics in biomedical research.</a> Nature. 507 (7491), 181-189 (2014).</li><li>Kim, S., Kim, H. J., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+applications+of+microfluidic+gradient+devices.">Biological applications of microfluidic gradient devices.</a> Integr Biol. 2 (11-12), 584-603 (2010).</li><li>Irimia, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+system+for+measuring+neutrophil+migratory+responses+to+fast+switches+of+chemical+gradients.">Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients.</a> Lab Chip. 6 (2), 191-198 (2006).</li><li>Lin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+migration+in+opposing+chemoattractant+gradients+using+microfluidic+chemotaxis+devices.">Neutrophil migration in opposing chemoattractant gradients using microfluidic chemotaxis devices.</a> Ann Biomed Eng. 33 (4), 475-482 (2005).</li><li>Ambravaneswaran, V., Wong, I. Y., Aranyosi, A. J., Toner, M., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+decisions+during+neutrophil+chemotaxis+inside+bifurcating+channels.">Directional decisions during neutrophil chemotaxis inside bifurcating channels.</a> Integr Biol. 2 (11-12), 639-647 (2010).</li><li>Jones, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+neutrophil+migration+patterns+during+sepsis+after+major+burns.">Spontaneous neutrophil migration patterns during sepsis after major burns.</a> PloS One. 9 (12), e114509 (2014).</li><li>Butler, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burn+injury+reduces+neutrophil+directional+migration+speed+in+microfluidic+devices.">Burn injury reduces neutrophil directional migration speed in microfluidic devices.</a> PloS One. 5 (7), e11921 (2010).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+platform+for+evaluating+neutrophil+chemotaxis+induced+by+sputum+from+COPD+patients.">A microfluidic platform for evaluating neutrophil chemotaxis induced by sputum from COPD patients.</a> PloS One. 10 (5), e0126523 (2015).</li><li>Sackmann, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+kit-on-a-lid:+a+versatile+platform+for+neutrophil+chemotaxis+assays.">Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays.</a> Blood. 120 (14), e45-e53 (2012).</li><li>Agrawal, N., Toner, M., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+migration+assay+from+a+drop+of+blood.">Neutrophil migration assay from a drop of blood.</a> Lab Chip. 8 (12), 2054-2061 (2008).</li><li>Jones, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+platform+for+measuring+neutrophil+chemotaxis+from+unprocessed+human+whole+blood.">Microfluidic platform for measuring neutrophil chemotaxis from unprocessed human whole blood.</a> J Vis Exp. (88), (2014).</li><li>Jones, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+assay+for+precise+measurements+of+mouse,+rat,+and+human+neutrophil+chemotaxis+in+whole-blood+droplets.">Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets.</a> J Leukocyte Biol. 100 (1), 241-247 (2016).</li><li>Sackmann, E. K. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+asthma+from+a+drop+of+blood+using+neutrophil+chemotaxis.">Characterizing asthma from a drop of blood using neutrophil chemotaxis.</a> P Natl Acad Sci. 111 (16), 5813-5818 (2014).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+all-on-chip+method+for+testing+neutrophil+chemotaxis+induced+by+fMLP+and+COPD+patient's+sputum.">An all-on-chip method for testing neutrophil chemotaxis induced by fMLP and COPD patient's sputum.</a> Technology. 04 (02), 104-109 (2016).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="581">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:581px;">Device fabrication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mask aligner</td>
			<td>ABM</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spinner</td>
			<td>Solitec</td>
			<td>5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>VWR</td>
			<td>11301-022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacuum dessicator</td>
			<td>Fisher Scientific</td>
			<td>08-594-15A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital scale</td>
			<td>Ohaus</td>
			<td>CS200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 2000 thinner</td>
			<td>Microchem</td>
			<td>SU-8 2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 2025 photoresist</td>
			<td>Microchem</td>
			<td>SU-8 2025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>Microchem</td>
			<td>SU-8 developer</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Si wafer</td>
			<td>Silicon, Inc</td>
			<td>LG2065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropyl alcohol</td>
			<td>Fisher Scientific</td>
			<td>A416-4</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:159px;">(tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane</td>
			<td>Gelest</td>
			<td>78560-45-9</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Polydimethylsiloxane 
			(PDMS)</td>
			<td>Ellsworth Adhesives</td>
			<td>2065622</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875714</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cutting pad</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Punchers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">Name </td>
			<td>Source</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">On-chip cell isolation and chemotaxis assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">RPMI 1640</td>
			<td>Fisher Scientific</td>
			<td>SH3025502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS</td>
			<td>Fisher Scientific</td>
			<td>SH3002802</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Bovine serum albumin 
			(BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>SH3057402</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Fibronectin</td>
			<td>VWR</td>
			<td>CACB356008</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">fMLP</td>
			<td>Sigma-Aldrich</td>
			<td>F3506-10MG</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Magnetic disks</td>
			<td style="width:124px;">Indigo Instruments</td>
			<td>44202-1</td>
			<td style="width:164px;">5 mm in diameter, 
			1 mm thick</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD10S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"> Rhodamine</td>
			<td> 
			Sigma-Aldrich</td>
			<td>R4127-5G</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Giemsa stain solution</td>
			<td style="width:124px;">Rowley Biochemical Inc.</td>
			<td style="width:135px;">G-472-1-8OZ</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:159px;">EasySep Direct Human 
			Neutrophil Isolation 
			Kit</td>
			<td style="width:124px;">STEMCELL 
			Technologies Inc</td>
			<td>19666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:159px;">Dithiothreitol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:159px;"> Nikon Ti-U inverted fluorescent microscope </td>
			<td style="width:124px;">Nikon</td>
			<td>Ti-U</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Microscope environmental chamber.</td>
			<td style="width:124px;">InVivo Scientific</td>
			<td>N/A</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:159px;"> CCD camera</td>
			<td style="width:124px;">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...

Watch this Scientific Journal Video about An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood at JoVE.com

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. 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.

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

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...