This work is sponsored by the Lupus Research Institute (NY, NY), NIH P01-AR49084, NIH R21-DA026956 and NIH UL1-TR00165. We thank Dr. Robert P. Kimberly for his continued support.

All donors recruited for this study gave written informed consent and the study was approved by the University of Alabama at Birmingham Institutional Review Board.
1. HUVEC Initial Culture and Subculture
<ol>
	<li>Culture human umbilical vein endothelial cells (HUVEC) in vitro in complete growth medium that is comprised of endothelial cell basal medium (see List of Materials) supplemented with endothelial cell growth factors. 
	Note: The growth factors used in this study include: 5 ng/ml human recombinant Epidermal Growth Factor (hEGF), 1.0 mg/ml hydrocortisone, 50 mg/ml Gentamicin and 50 ng/ml amphotericin-B (GA-1000), 2% Fetal Bovine Serum (FBS), 0.5 ng/ml Vascular Endothelial Growth Factor (VEGF), 10 ng/ml human Fibroblast Growth Factor-Basic with heparin (hFGF-B), 20 ng/ml human recombinant Insulin-like Growth Factor (R3-IGF-1), 1 &#181;g/ml ascorbic acid, and 22.5 &#181;g/ml heparin. The complete medium is prepared initially at 37 &#176;C and can then be stored at 4 &#176;C for use within 1&#160;month of preparation.</li>
	<li>To prepare a flask for cells, complete growth medium is added to a 75 cm2 tissue culture flask (1 ml/5 cm2) and then the flask is allowed to equilibrate to 37 &#176;C/5% CO2 in a humidified incubator for at least 30 min. Human HUVEC will be seeded directly into this equilibrated culture flask at a density of 2,500-5,000 cells/cm2.</li>
	<li>While the media is equilibrating, quickly thaw the stock HUVEC cryovial in a 37 &#176;C water bath. Disperse the cells in the original storage vial by vortexing and then add them directly to the culture flask containing the pre-equilibrated HUVEC complete growth medium to achieve a density of 2,500-5,000 cells/cm2. Gently rock the flask to evenly distribute the cells and then return the flask to the incubator. These cells now represent the 1st passage.</li>
	<li>Medium should be changed every two days until the cells are 70-80% confluent.</li>
	<li>The HUVECs can now be harvested from the culture flask with Trypsin/EDTA. The culture media is aspirated from the culture flasks followed by a PBS rinse to remove any residual protein and calcium from the cells. A 0.25% Trypsin-EDTA solution is added and within 2-6 min cell detachment should be evident as assessed by light microscopy.</li>
	<li>When 90% of the cells are rounded up off the plate, stop the trypsinization by adding an equal volume of 2x trypsin inhibitor. To facilitate the harvest of cells, add another equal amount of complete growth medium. Transfer the detached cells to a sterile 15 ml centrifuge tube.</li>
	<li>Centrifuge the detached cells at 225 x g for 5 min at room temperature. Aspirate the supernatant, and then resuspend the cells in 2-3 ml of complete growth medium. Determine the cell concentration and viability using a hemacytometer and Trypan Blue.</li>
	<li>To utilize the harvested cells for study, re-seed additional 75 cm2 flasks with cells at a density of 10,000 cells/cm2 and proceed to step 2.2. Alternatively, cells can be frozen at this point for future studies. For cell freezing, pellet the cells by centrifugation at 225 x g for 5 min at room temperature. Aspirate off the supernatant and resuspend the cell pellet in FBS containing 10% DMSO at a concentration of 1 x 106 cells/ml. The cell suspension is then transferred to cryovials and stored at -80 &#176;C after freezing of the cells in a cell-freezing container.</li>
</ol>
2. HUVEC Layer Preparation
<ol>
	<li>Use of frozen 2nd passage HUVECs: revival and culture. If using actively growing cells, proceed to step 2.2.
	<ol>
		<li>Thaw the cryovial containing 2nd passage HUVECs from step 1.8 quickly in a 37 &#176;C water bath. Transfer cells from the cryovial to a 15 ml sterile centrifuge tube and add 10 ml growth medium.</li>
		<li>Centrifuge the cells at 200 x g for 5 min at room temperature and aspirate the supernatant to remove the residual DMSO.</li>
		<li>Resuspend the cell pellet with complete growth medium and transfer the cells into a 75 cm2 flask. Add growth medium to a total volume about 20-25 ml and incubate at 37 &#176;C/5% CO2.</li>
	</ol>
	</li>
	<li>Visually examine the cell culture for confluence each day with media changes every two days. Usually within 2-4 days, cells will reach 80-90% confluence at which time they will be ready for use in the flow chamber assay.</li>
	<li>To prepare the culture dish that will be used in the flow chamber (see Section 5), add 1 ml of 10 &#181;g/ml fibronectin and 0.05% (w/v) gelatin to a 35 mm tissue culture dish and pipette several times to make sure the whole plate surface is coated. Remove the excessive fibronectin and gelatin solution and air dry the plate for at least 30 min to optimize the protein matrix formation. The fibronectin and gelatin solution may be reused up to 10x.</li>
	<li>Harvest the HUVEC cells from step 2.2 using trypsin-EDTA as described in steps 1.5-1.7. Seed 500,000 cells into each coated tissue culture dish. Add 2 ml growth medium in each dish and incubate at 37 &#176;C/5% CO2.</li>
	<li>Allow cells to grow to confluence as in step 2.2. Cells should be visually inspected daily with medium changes every two days. Prior to the flow chamber assay, prime the HUVEC with 20 ng/ml human TNF-&#945; for 4-6 hr to upregulate and stimulate adhesion molecule expression.</li>
</ol>
3. Purified Ligand Coating
<ol>
	<li>Draw a circle of 0.5 cm diameter with a marker or pen at the center of a 35 mm tissue culture dish.</li>
	<li>Plate 20 &#181;l of 20 &#181;g/ml protein A in the marked area. Use the pipette tip to spread the protein A solution to cover the whole area within the 0.5 cm diameter circle. It is important to not touch or scratch the surface of the dish. Incubate at 37 &#176;C for 1 hr.</li>
	<li>Wash the protein-A coated plate 3x&#160;with 1 ml of PBS (pH 8.0).</li>
	<li>Plate 50 &#181;l 1% BSA in the marked area to block non-specific binding on the plate. Incubate for 2 hr at 4 &#176;C.</li>
	<li>Wash the blocked plate 3x&#160;with 1 ml of PBS as in step 3.3.</li>
	<li>Prepare the Fc-adhesion receptor ligand chimeric protein solutions for coating. In this experiment, an ICAM-1/Fc chimera solution at 25 &#181;g/ml and a P-Selectin/Fc chimera at 0.5 &#181;g/ml in PBS (pH 8.0) was used.</li>
	<li>Coat the marked area with 50 &#181;l of substrate. Incubate overnight at 4 &#176;C. The dish should be used within two days and the coated area should not be allowed to dry out. Add PBS if necessary to maintain the 50 &#181;l solution on the plate.</li>
</ol>
4. Neutrophil Separation (All Steps Performed at Room Temperature)
<ol>
	<li>After obtaining informed consent, collect participant blood by phlebotomy into an anticoagulant blood collection tube or vacutainer (EDTA or heparin). After blood collection, dilute the blood 1:1 with PBS before separation.</li>
	<li>Prepare a two layer Ficoll for separating PBMC and neutrophils in 50 ml centrifuge tubes. First add 15 ml heavy Ficoll (see List of Materials, &#961;=1.118-1.120), and then carefully layer 10 ml light Ficoll (see List of Materials, &#961;=1.077-1.080) on top of the heavy Ficoll. There should be a sharp border between the light Ficoll and heavy Ficoll layers. Finally, carefully layer 25 ml of the diluted blood sample on top of the light Ficoll without disturbing the Ficoll layer.</li>
	<li>Centrifuge the tube at 250 x g for 30 min at room temperature. Note: The centrifuge rotor brake should be off for these spins to minimize potential disruption of the cell separation during rotor deceleration at the end of the centrifugation. After centrifugation, the following layers (from top to bottom) are present: the top layer is the plasma followed by the PBMC layer on top of the light Ficoll layer, the neutrophil layer with few red blood cells (RBC) is between the light and heavy Ficoll followed by the heavy Ficoll layer and RBCs at the bottom of the tube.</li>
	<li>Harvest and transfer the neutrophil layer into a new 50 ml tube with a transfer pipette, add PBS to a final volume of 50 ml and centrifuge at 225 x g for 10 min at room temperature.
	<ol>
		<li>After centrifugation, there may still be RBCs mixed with the neutrophils. Aspirate the supernatant down to the 10 ml mark. Resuspend the neutrophil-RBC pellet by gentle agitation of the tube (or a brief low speed vortexing) and then wash again with 50 ml of PBS.</li>
	</ol>
	</li>
	<li>After the second wash, aspirate the supernatant. To remove the contaminating RBCs, resuspend the pelleted cells in the residual PBS by agitation (or short vortexing), add 25 ml H2O and gently vortex for 10 sec to lyse the RBC.
	<ol>
		<li>Add 25 ml of 1.8% NaCl and immediately mix by centrifugation at 225 x g for 10 min at room temperature. The contaminating RBC should now be lysed leaving a white neutrophil cell pellet.</li>
	</ol>
	</li>
	<li>Wash the neutrophil cell pellet with PBS.</li>
	<li>Resuspend the isolated neutrophils in RPMI medium with 10% FBS and determine the cell concentration under light microscope with a hemocytometer.</li>
	<li>Adjust the cell density to 500,000 cells/ml with complete RPMI-10% FBS medium.</li>
</ol>
5. Flow Chamber Adhesion Assay
<ol>
	<li>
	Assemble the flow chamber. Place the 35 mm dish containing the confluent HUVECs or purified adhesion receptor ligands on the microscope table. Connect the parallel plate flow chamber with syringe pump, vacuum system and leave one line open for the neutrophil input. Insert the flow chamber on top of the plate and fasten the flow chamber assembly13. (See Figure 1)
	</li>
	<li>Start the video recording program on the computer connected to the microscope. Adjust the field and focus of the microscope until a clear field with fully grown HUVEC cells or a field within the ligand coating region is visible.</li>
	<li>Using the syringe pump, rinse the flow chamber with RPMI medium. Make sure there are no air bubbles within the chamber or the neutrophil input line.</li>
	<li>If desired, the neutrophils may be primed with low dose (10-8 M) fMLP for 10 min. This will allow for a matching of the basal level of neutrophil activation between different donors14.</li>
	<li>Use the syringe pump to inject the neutrophils into the flow chamber at defined speeds (a speed of 350 &#181;l/min, which equals a shear stress of 1.5 dynes/cm2 is used in this study). Record the video. Because neutrophil adhesion can occur rapidly, a video with a length of 4-5 min is usually sufficient to quantitate adhesion events for analysis.</li>
	<li>An adherent cell is defined as a cell that moves less than one cell diameter within 5 sec on the HUVEC or ligand coated surface15,16. Count the total number of adherent cells in the field present in the recorded video using this rule. By recording similar length videos with different donors&#8217; neutrophil, one can calculate the adherent cells/min to compare the adhesion properties between different donors.</li>
</ol>

<ol>
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Diseases. 71 (11), 1809-1814 (2012).</li><li>Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+leukocyte+recruitment+in+the+inflammatory+process.">Molecular mechanism of leukocyte recruitment in the inflammatory process.</a> Cardiovasc. Res. 32 (4), 733-742 (1996).</li><li>Korthuls, R. J., Anderson, D. C., Granger, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+neutrophil-endothelial+cell+adhesion+in+inflammatory+disorders.">Role of neutrophil-endothelial cell adhesion in inflammatory disorders.</a> J. Crit. Care. 9 (1), 47-71 (1994).</li><li>Albelda, S. M., Smith, C. W., Ward, P. 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The authors have nothing to disclose.

This protocol guides the separation and isolation of minimally activated neutrophils for the careful quantification of neutrophil adhesion under sheer stress conditions. Neutrophil adhesion is a critical process in inflammation. Because genetic variants in multiple molecules in this process have been demonstrated to predispose to the development of autoimmune disease1,2, an assay system capable of quantitatively assessing human neutrophil firm adhesion is required. The method described in this protocol allows for the careful and quantitative determination of the firm adhesive potential of neutrophils in a controlled in vitro environment under sheer stress. This method thus allows the direct comparison of quantitative neutrophil adhesion between genotyped individuals to determine the importance of genetic variation in adhesion molecules14.
Several steps in this method merit careful consideration to achieve highly quantitative and reproducible results. In the HUVEC preparation, it is critical to attain 100% cell confluence before using them in the flow chamber. For using surfaces coated with purified ligand, the substrate coating area should never be allowed to dry out to avoid denaturing the ligand. In addition, the preparation of human neutrophils is critical to the success of the experiment. Key issues in isolating neutrophils from blood include gently handling by minimizing vortexing to avoid activation, keeping the cells at room temperature (i.e. the blood should not be stored on ice and centrifugation steps should be performed at room temperature) and completing the isolation and the experiment in the least amount of time as possible. There are additional neutrophil isolation methods that can also be utilized to prepare cells for these assays17,18.
From a practical perspective, adhesion assays using freshly isolated human neutrophils should be initiated within 3-6 hr&#160;after participant phlebotomy. As neutrophils are extremely sensitive to handling, prolonged times between the blood draw and usage could affect assay results. Careful determination of neutrophil cell concentration prior to the flow chamber assay is also necessary to achieve accurate and reproducible results.
During the flow chamber assay, it is important to monitor the video recording carefully to ensure that the flow speed is consistent and there is no turbulence throughout the length of the experiment. Changes in flow speeds or the presence of turbulence would necessitate that the experiment be repeated. After the experiment, it is also important to assess the remaining neutrophils microscopically to ensure that the neutrophils are not clumping. Clumping at this point would indicate that the neutrophils have become activated which could significantly alter neutrophil density during the experiment.
The flow chamber creates a near homogeneous sheer stress within the chamber (&#964;=6Q&#956;/(wh2), where Q=flow rate, &#956;=dynamic viscosity, and w=width of the flow chamber, h=height of the flow chamber15). In our studies, we used a flow rate of 350 &#181;l/min for neutrophil adhesion, which creates a sheer stress of 1.5 dynes/cm2 (w=0.25 cm, h=0.01 in., the viscosity of water at 37 &#176;C (0.007 poise) was used as an approximation of the viscosity of RMPI media). For a specific flow chamber, one can change the flow rate to achieve different levels of sheer stress to mimic different physiological conditions. Typical physiologic shear stress in human blood vessels can ranges between 0.5-5.0 dynes/cm 13,16. Shear stress in other blood vessels and other animals were listed in Table 1113,16,19&#160;20.
While our method has focused on the study of the adhesion of human neutrophils, this method is not limited to neutrophils and can easily be applied to adhesion or rolling studies of other cell types with simple modifications. Also, the substrates in this method can be changed for different purposes.
Although this protocol is easily adaptable to different studies, there are some limitations. The protocol as implemented here requires large number of primary cells for analysis. This may preclude analysis of primary cells from small animals. Additionally, the need to immobilize purified adhesion ligands in an active/available conformation may limit the range of ligands. The use of Fc-fusion proteins greatly enhances the chances of achieving proper ligand conformation on the plate surface. Nonetheless, our method has significant flexibility to allow the quantitative analysis of adhesion events. These studies will greatly enhance our understanding of adhesion receptor-ligand pairs, and the potential function importance of genetic variants in these proteins, in the pathogenesis of human diseases.

Genetic variants in both &#946;2 integrins and in ICAM ligands are now recognized to be associated with the development of autoimmune disease1,2. The determination of the functional consequences of these variants in cells derived from individuals with these variants is necessary for our understanding of how these variants contribute to autoimmune disease pathogenesis. Such functional studies allow for the determination of the mechanisms by which naturally occurring genetic variants shape the immune response in both health and disease. In the specific example of SLE, we now know that variants in ITGAM (CD11b) and its ligand, ICAM-1, strongly associate with development of disease1,2. Because neutrophils are critical in inflammatory responses, the quantitative study of neutrophil adhesion may provide mechanistic insights into how genetic variants in ITGAM/ICAM alter inflammation.
Neutrophil firm adhesion to endothelial cells (EC) is a highly regulated process and plays an essential role in inflammatory responses3,4. The firm adhesion of neutrophil follows initial neutrophil rolling and capture on the EC and ultimately can result in transmigration in vivo. These processes involve many different kinds of adhesion molecules, including ICAM-1, ICAM-2, P-Selectin, E-Selectin on the endothelial cells and &#946;2 integrins on the neutrophil5-9. Thus, careful quantification of neutrophil adhesion from donors with different allelic variants of adhesion molecules will be important to understand the functional and pathological consequences of these genetic variants.
Experimental use of a flow chamber can create an in vitro environment with fluid shear stress similar to that observed in the blood vessel environment in vivo10-12. Indeed, a flow chamber assay coupled with human umbilical vein endothelial cell (HUVEC) can mimic the in vivo environment of a blood vessel. Using this method, one can study the overall cellular adhesive properties towards endothelial cell. In addition, the highly controlled environment of the flow chamber also allows assessment of cell binding to purified adhesion ligands such as ICAM-1 to facilitate the study of specific receptor-ligand interactions.
We present here a method utilizing a flow chamber adhesion assay system to study the adhesive properties of human peripheral blood neutrophils to HUVEC and to purified ligand substrates. Using this method with cells from donors expressing different adhesion molecule allelic variants allows us to assess how these genetic variants can alter human neutrophil firm adhesion.

<table border="0" cellpadding="0" cellspacing="0" style="width:677px;" width="677">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:677px;">Table of Reagents Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Name of reagent</td>
			<td style="width:107px;">Company</td>
			<td style="width:111px;">Catalogue number</td>
			<td style="width:268px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">0.25% Trypsin/EDTA</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">25200-056</td>
			<td style="width:268px;">with Phenol Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">2X Trypsin inhibitor</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">R-002-100</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">35mm tissue culture dish</td>
			<td style="width:107px;">Corning Inc.</td>
			<td style="width:111px;">430165</td>
			<td>Standard Tissue Culture Treated Surface</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:192px;">75cm2 tissue culture flask</td>
			<td style="width:107px;">Corning Inc.</td>
			<td style="width:111px;">430641</td>
			<td>Standard Tissue Culture Treated Surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">CCD camera</td>
			<td style="width:107px;">Dage-MTI</td>
			<td style="width:111px;">Model 300T-RC</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Cell freezing container&#160;</td>
			<td style="width:107px;">Biocision</td>
			<td style="width:111px;">BCS-045</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">EBM-2</td>
			<td style="width:107px;">Lonza Inc.</td>
			<td style="width:111px;">CC-3156</td>
			<td style="width:268px;">HUVEC growth basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">EGM-2 Bulletkit</td>
			<td style="width:107px;">Lonza Inc</td>
			<td style="width:111px;">CC-4176</td>
			<td style="width:268px;">HUVEC growth factors for basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">FBS</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">10082-147</td>
			<td style="width:268px;">Certified, Heat Inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Gelatin</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">G9391</td>
			<td style="width:268px;">from bovine skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Hemacytometer</td>
			<td style="width:107px;">Fisher scientific</td>
			<td style="width:111px;">02-671-51B&#160;</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Fibronectin</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">F2006</td>
			<td style="width:268px;">from human plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Flow chamber</td>
			<td style="width:107px;">Glyco Tech</td>
			<td style="width:111px;">31-001</td>
			<td style="width:268px;">Circular flow chamber for 35mm dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">fMLP</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">47729</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Histopaque-11191</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">11191</td>
			<td style="width:268px;">Heavy ficoll</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">HUVEC</td>
			<td style="width:107px;">Lonza Inc.</td>
			<td style="width:111px;">CC-2517A</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">ICAM-1</td>
			<td style="width:107px;">R&#38;D systems</td>
			<td style="width:111px;">720-IC</td>
			<td style="width:268px;">Fc chimera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Lymphocyte separation medium</td>
			<td style="width:107px;">Mediatech Inc.</td>
			<td style="width:111px;">25072CV</td>
			<td style="width:268px;">Light ficoll</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Microscope</td>
			<td style="width:107px;">Zeiss</td>
			<td style="width:111px;">Axiovert 100</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">RPMI 1640 medium</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">11875</td>
			<td style="width:268px;">with L-Glutamine and Phenol Red&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Protein A</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">P6031</td>
			<td style="width:268px;">resuspend in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">P-Selectin</td>
			<td style="width:107px;">R&#38;D systems</td>
			<td style="width:111px;">137-PS</td>
			<td style="width:268px;">Fc chimera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Syringe pump</td>
			<td style="width:107px;">KD Scientific</td>
			<td style="width:111px;">KDS270</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">TNF-a</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">PHC3015</td>
			<td style="width:268px;">Recombinant Human Protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:107px;">Life technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Examples of neutrophil binding to a purified ligand (ICAM-1/P-Selectin) coated flow chamber (Figure 2) or of neutrophil binding to a HUVEC coated flow chamber assay (Figure 3) are shown. As shown in the figures, neutrophils continue to accumulate/adhere to the coated surface or HUVEC under conditions of continuous flow. Under our typical experiment conditions, we can observe 50-70 human neutrophils firmly adhering to the coated surface or HUVEC during a four-minute recording period. However, allelic variants of neutrophil adhesion molecules, or allelic variants in the substrate (purified ligand or HUVEC), could substantially alter quantitative neutrophil adhesion14.
We have also assessed the time dependence of the neutrophil adhesion events observed in our studies. While we are maintaining constant flow conditions, it is possible that the adhesive properties of the cells change over time. However, over the relatively short time courses in our studies, we do not observe any consistent differences in the rate of adhesion over time. For example, assessing adhesion at the 1-2 minute time points compared to adhesion observed between the 3-4 min time points are not consistently different. Of course, if cells are being stimulated during the adhesion experiment, then changes in adhesive properties over time could be expected.
In our studies, we controlled for isolation induced neutrophil activation by intentionally priming the cells with low dose fMLP (10-8 M) for 10 min prior to study. Endothelial cells (HUVEC in our studies) also require prior activation to upregulate adhesion molecule expression for optimal leucocyte firm adhesion. In the absence of pre-treatment, endothelial cells (HUVECs) will support very little neutrophil adhesion. In our studies, we used 10 ng/ml TNF&#945; treatment for 4-6 hr prior to use. IL-1&#946; (10 ng/ml) and LPS (0.5-1 &#181;g/ml) can also be used to pre-activate the endothelial cells. Importantly, untreated HUVEC can be used as negative control to ensure that cell adhesion is caused by specific (receptor-mediated) neutrophil-endothelial cell interactions. Alternatively, anti-receptor antibodies can be used to block specific receptors to assess specificity of adhesion.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51410/51410fig1highres.jpg" src="/files/ftp_upload/51410/51410fig1.jpg" /> 
Figure 1. Flow chamber configuration on the microscope stage. <a href="https://www.jove.com/files/ftp_upload/51410/51410fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51410/51410fig2highres.jpg" src="/files/ftp_upload/51410/51410fig2.jpg" /> 
Figure 2. Screen shots from a sample video of neutrophils adhering to ICAM-1/P-Selectin coated surface at different time points (A: 0 time point, B: 1 min time point, C: 2 min time point, and D: 4 min time point). The concentration of ICAM-1 is 25 &#181;g/ml and P-Selectin is 0.5 &#181;g/ml. Neutrophil flow speed is 350 &#181;l/min with neutrophil density at 500,000 cells/ml.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/51410/51410fig3highres.jpg" src="/files/ftp_upload/51410/51410fig3.jpg" /> 
Figure 3. Screen shots from a sample video of neutrophils adhering to HUVEC coated surface at different time points (A: 0 time point, B: 1 min time point, C: 2 min time point, and D: 4 min time point). Neutrophil flow speed is 350 &#181;l/ml with neutrophil density at 500,000 cells/ml.
<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td>Species</td>
			<td >Location</td>
			<td >Shear stress (dynes/cm2)</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Common caroid artery</td>
			<td>11.6</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Branchial artery</td>
			<td>6.5</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Common femoral artery</td>
			<td>4.3</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Suprarenal aorta</td>
			<td>7.3</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Supraceliac aorta</td>
			<td>4.2</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Superficial fermoral artery</td>
			<td>4.4</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Venules</td>
			<td>0.5-5.0</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Retinal first arterioles </td>
			<td>40.2</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Retinal second arterioles </td>
			<td>0.001</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Retinal first venules</td>
			<td>23.2</td>
		</tr>
		<tr >
			<td>Human</td>
			<td>Retinal second venules</td>
			<td>0.43</td>
		</tr>
		<tr >
			<td>Dog</td>
			<td>Common caroid artery</td>
			<td>15.8</td>
		</tr>
		<tr >
			<td>Rabbit</td>
			<td>Common caroid artery</td>
			<td>23.3</td>
		</tr>
		<tr >
			<td>Rat</td>
			<td>Common caroid artery</td>
			<td>46.6</td>
		</tr>
		<tr >
			<td>Mouse</td>
			<td>Common caroid artery</td>
			<td>64.8</td>
		</tr>
		<tr >
			<td>Dog</td>
			<td>Common femoral artery</td>
			<td>9.8</td>
		</tr>
		<tr >
			<td>Rabbit</td>
			<td>Common femoral artery</td>
			<td>156.8</td>
		</tr>
		<tr >
			<td>Rat</td>
			<td>Common femoral artery</td>
			<td>65.9</td>
		</tr>
	</tbody>
</table>
Table 1. Sample shear stress in different organs and different species.
* summarized from references 13, 16, 19, and 20.

Watch this Scientific Journal Video about Human Neutrophil Flow Chamber Adhesion Assay at JoVE.com

Human Neutrophil Flow Chamber Adhesion Assay

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 ...

human-neutrophil-flow-chamber-adhesion-assay

Research

JoVE Journal

Immunology and Infection

16.8K 조회수.  University of Alabama at Birmingham.  호중구의 부착을 정량하는 방법이보고되어있다. 이 방법은 혈관에서 발생 된 것과 유사한 동적 흐름 환경을 생성한다. 그것은 허용 정제 부착 분자 (리간드) 또는 투명 스트레스 생체 환경 유사한 맥락에서 내피 세포 기질 (HUVEC) 중 하나에 호중구 부착 조사. 

비디오: Human Neutrophil Flow Chamber Adhesion Assay

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

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 ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...