This work was supported in part by grants from National Institutes of Health (P30 HL101302 and RO1 HL109439 to J.C.) and American Heart Association (SDG 5270005 to J.C.). A. Barazia was supported by a T32HL007829 NIH training grant.

1. Preparation of Intravital Microscope (Figure 1A)
<ol>
	<li>Prepare superfusion buffer (125 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, and 17 mM NaHCO3, pH 7.4).</li>
	<li>Turn on a circulatory water bath to maintain temperature of buffer and a thermo-controlled blanket at 37 &deg;C. Aerate buffer with nitrogen gas (5% CO2 balanced with nitrogen).</li>
	<li>Turn on the microscope system (Sutter Lambda DG-4 high speed wavelength changer, workstation computer, Olympus BX61W microscope, MPC-200 multi-manipulator, ROE-200 stage controller, high speed camera, and intensifier).</li>
	<li>For the vascular inflammation model, use a 100% dichroic mirror. To induce thrombus formation by laser injury, replace a 100% mirror with a 50/50% mirror and turn on a micropoint laser ablation system.</li>
</ol>
2. Preparation of Cremaster Muscle for Intravital Microscopy (Figure 1B)
<ol>
	<li>Anesthetize a male mouse (6-8 weeks old, C57BL/6) by i.p. injection of ketamine (125 mg/kg body weight (BW)) and xylazine (12.5 mg/kg BW). The University of Illinois Institutional Animal Care and Use Committee approved all animal care and experimental procedures. For a vascular inflammation model, inject murine TNF-&alpha; (0.5 &mu;g in 250 &mu;l saline) intrascrotally into a mouse 3 hr prior to imaging (2-2.5 hr prior to surgery).</li>
	<li>Place the mouse on a thermo-controlled blanket at 37 &deg;C on an intravital microscope tray.</li>
	<li>Pull up the skin on the surface of the neck using forceps and incise horizontally the midline of the neck skin up to 1 cm with scissors.</li>
	<li>Gently open the cervical muscle using blunt scissors and remove the muscle surrounding the trachea.</li>
	<li>Cannulate a PE90 tube into trachea to eliminate breathing difficulty.</li>
	<li>Gently remove the left side of cervical muscle and isolate the jugular vein from surrounding tissues.</li>
	<li>Cannulate a PE10 tube into the left jugular vein for infusion of antibodies and additional anesthetics.</li>
	<li>Gently pull out and incise the scrotal skin horizontally. To maintain tissue integrity throughout the experiment, prewarmed buffer was superfused over the surgical field.</li>
	<li>Press on the lower abdomen with one forcep and carefully pull out a testicle with the other forcep.</li>
	<li>Remove the surrounding connective tissues around the testis.</li>
	<li>Incise the cremaster muscle vertically and flatten the muscle over a glass coverslip on an intravital microscope tray by pinning the periphery.</li>
	<li>Allow 10-20 min for the muscle to stabilize prior to data collection. Cremaster muscle arterioles (for thrombus formation) and venules (for vascular inflammation) with a diameter of 30-45 &mu;m were chosen for our study. </li>
	<li>To maintain anesthetic conditions, infuse 30-50 ml of the same anesthetic approximately every 30 min through the jugular cannulus.</li>
</ol>
3. Intravital Microscopy for TNF-&alpha;-induced Vascular Inflammation
<ol>
	<li>After placement of the mouse on the intravital microscope, open SlideBook 5.0 software and click on "Focus Window" on menu bar to set the appropriate optics and objective (60X).</li>
	<li>Infuse Dylight 488-conjugated rat anti-mouse CD42c (0.1 &mu;g/g BW in 100 &mu;l saline) and Alexa Fluor 647-conjugated rat anti-mouse Gr-1 antibodies (0.05 &mu;g/g BW in 100 &mu;l saline) through a jugular cannulus.</li>
	<li>Go to menu bar, and click on "Image Capture" in the toolbar.</li>
	<li>A new menu will pop up; In "Image", select "Bin Factor" as 1x1 and select "640" on width and "460" on height.</li>
	<li>In "Filter Set", mark the checkbox for the channels that you would like to expose and set the exposure time for each. For example, open channel: 10 msec, FITC channel: 50 msec, and Cy5 channel: 50 msec.</li>
	<li>Select "Time-Lapse Capture" to adjust number of time points and interval. For the inflammation model, the number of time points is set to 1,000-1,500 with an interval of 200 msec (Total elapsed time: 5 min).</li>
	<li>Click "Test" to see a single-plane image at the specified exposure time for that channel. Adjust exposure time and/or microscope light until the histogram shows an adequate intensity distribution (For multi-channel capture, repeat this process for each channel).</li>
	<li>Once all of the parameters are set (channels, exposure times, and so on) in the "Image Capture", select "Start" to begin capture.</li>
	<li>Monitor rolling and adherent platelets and neutrophils for 5 min in the top half of an inflamed cremaster venule in the vascular inflammation model. Eight to ten different venules are recorded in one mouse.</li>
	<li>Images were taken using a total magnification of 60X (1.0 NA water immersion objective) giving a window size of 157 &mu;m x 118 &mu;m.</li>
	<li>Upon completion of the experiment, the mice were euthanized via cervical dislocation.</li>
</ol>
4. Data Analysis for the Venular Inflammation Model
<ol>
	<li>Open SlideBook 5.0 software and click "Folder" on the menu bar to open a file.</li>
	<li>Click on the drop-down channel menu and select "Open, FITC, Cy5, etc."</li>
	<li>Click "Renormalize (red and green bars)" and select the channel. Drag the red and green bars to the left or right to roughly optimize the fluorescence signal.</li>
	<li>Click "Apply" and "OK" to update the image.</li>
	<li>Click "Thumbnail Button" to select current display as the default.</li>
	<li>Play captured timelapse image, and count rolling and adherent neutrophils during the 5 min period.</li>
	<li>To analyze platelet thrombus formation, go to "Mask" and select "Create".</li>
	<li>Name a mask and mark "In the current image".</li>
	<li>Click "Large pencil icon" in "Marquee Tool" in the main view.</li>
	<li>Color a region outside the vessel in the main view to set a background mask.</li>
	<li>Go to "Mask", click "Copy This Plane", click "Copy mask in current timepoint" and select "All timepoints", and click "OK".</li>
	<li>To calculate background signal, go to "Statistics" and click "Mask Statistics".</li>
	<li>Click "Current 2D Time Lapse or 4D Image(s)" in Image Scope, and select "Entire Mask" in Mask Scope. In Features, select "Elapsed Time (hh:mm:ms)" under Date of Capture, select "Area (pixels)" under Morphometry, select "Maximum Intensity" under Intensity, and click "Export" (This statistic method allows us to calculate auto-fluorescence intensity (background signal)).</li>
	<li>Open the text file in MS Office Excel and calculate the average value of background signal throughout the recording period. This is the background fluorescence intensity.</li>
	<li>To determine thrombus intensity, click "Large pencil icon" again in the "Marquee Tool" in the main view.</li>
	<li>Color inside the vessel in the main view to set platelet signal.</li>
	<li>Go to "Mask", click "Copy This Plane", click "Copy mask in current timepoint", select "All timepoints", and click "OK".</li>
	<li>Go to "Statistics" and click "Mask Statistics".</li>
	<li>Click "Current 2D Time Lapse or 4D Image(s)" in Image Scope, and select "Entire Mask" in Mask Scope. In Features, select "Elapsed Time (hh:mm:ms)" under Date of Capture, select "Area (pixels)" under Morphometry, select "Sum Intensity" under Intensity, and click "Export" (This statistic method allow us to calculate the fluorescence intensity inside the vessel).</li>
	<li>Open the text file in MS Office Excel. Fluorescence signal of platelets is calculated by subtracting the average value of background intensity x Area (pixel) from the sum intensity of the inside of the vessel. This is the fluorescence intensity of the platelet thrombus.</li>
	<li>Repeat this in 30 different venules in 3-5 different mice. Then, calculate median value of the fluorescence intensity of platelets.</li>
	<li>To analyze the neutrophil rolling and adhesion, the rolling influx of neutrophils was determined over 5 min in each venule and presented as cell numbers per minute. The number of adherent neutrophils that were stationary for &gt;30 sec and slowly crawled (&lt;10 &mu;m over 30 sec) but did not roll over, were counted and presented as cell numbers per 5 min.</li>
</ol>
5. Intravital Microscopy for Laser-induced Arteriolar Thrombosis
5-1 Calibration of ablation laser
<ol>
	<li>Place a mirrored slide on microscope stage, apply a small drop of distilled water to the top of the slide, and adjust the intensity and focus using the eyepiece.</li>
	<li>Open "Focus Window" and select the "FRAP" tab in SlideBook 5.0 software.</li>
	<li>Set the laser power at 40-50, and set both "Double click repetitions" and "Double click size" to 8. Mark on the "Guide" box to bring up a set of crosshairs.</li>
	<li>Select the "Arrow" icon from the taskbar on the top of the screen.</li>
	<li>Under the "FRAP Alignment" tab, click "Fire next". A small spot should appear near the lower left corner on the monitor. Once the spot appears, double click on the center of it. Once clicked, another spot should appear above and to the right of the first spot; double click on it. Repeat this for 16 points (the 16th spot will appear in the lower right corner.) Click "Save" when complete to update the calibration parameters.</li>
	<li>To test the accuracy of the calibration, click on the "Center" button-a small spot should appear in the center of the screen. If not, repeat the calibration until desired accuracy is achieved.</li>
</ol>
5-2 Laser-induced arteriolar thrombus formation
<ol>
	<li>Place an anesthetized mouse (see the procedure of 2-2 to 2-12) on the microscope stage.</li>
	<li>Under the "Capture" window, select a protocol or create a new one. The settings are as follows: CY5 (70 msec exposure), Open (10 msec exposure), and FITC (50 msec exposure). Image is captured over 20 min (approximately 2,400 time points with an interval time of 500 msec).</li>
	<li>Infuse Dylight 488-conjugated anti-mouse CD42c and Alexa Fluor 647-conjugated anti-mouse Gr-1 antibodies as described above.</li>
	<li>Optimize microscope parameters including fluorescence intensity and bright field image as described in 3-3 to 3-7.</li>
	<li>Click the "Test" button to ensure proper brightfield intensity or antibody signal, arteriole placement and focus.</li>
	<li>Click "Start" to initiate the image capture process.</li>
	<li>Before firing the ablation laser, make sure that the mouse pointer icon has been selected and that the vessel wall is in clear focus.</li>
	<li>To fire the laser, double click on a spot 2-3 &mu;m internally from the vessel wall. Injury of the arteriolar endothelial cells causes a visual shape change that should be apparent in the brightfield image, followed by platelet accumulation. Monitor thrombus formation for 5 min after laser injury.</li>
	<li>Five minutes after laser injury (the thrombus size should now be stable) pause and cancel the capture. Subsequently, start a capture with 2400 time points with a 500 msec interval to record adherent platelets and rolling/adherent neutrophils for 20 min. Three to five arterioles are recorded in one mouse.</li>
	<li>Upon completion of the experiment, the mice were euthanized via cervical dislocation.</li>
</ol>
6. Data Analysis for the Arteriolar Thrombosis Model
<ol>
	<li>Go over steps 4.1 through 4.5.</li>
	<li>To obtain the background fluorescence intensity during platelet thrombus formation, go over steps 4.7 through 4.14.</li>
	<li>Go to "Mask", click "Segment", select FITC in channel, and insert the average value of background signal on Low. Click "Apply" and "OK".</li>
	<li>To analyze the fluorescence intensity of platelet thrombus (Dylight 488-conjugated anti-CD42c antibody), go over steps 4.18 through 4.19. To calculate the antibody signal, "Sum Intensity" is subtracted by the background signal ("Maximum Intensity" x "Area (pixels)"). This is the fluorescence intensity of the platelet thrombus.</li>
	<li>To quantify the rolling and adherent neutrophils, play the timelapse and count the number of cells that visibly roll over the platelet thrombus over 20 min. Rolling is defined as a decrease in neutrophil speed while interacting with the platelet thrombus for at least 2 sec. Adherent neutrophils are defined as any neutrophils that remain attached to the platelet thrombus for at least 2 min. The number of rolling and adherent neutrophils was presented as cell numbers per 20 min. Rolling velocity was calculated using a particle tracking system.</li>
</ol>

<ol>
	<li>Wagner, D. D., Frenette, P. 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+vessel+wall+and+its+interactions.">The vessel wall and its interactions.</a> Blood. 111, 5271-5281 (2008).</li><li>Nieswandt, B., Kleinschnitz, C., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischaemic+stroke:+a+thrombo-inflammatory+disease.">Ischaemic stroke: a thrombo-inflammatory disease.</a> J. Physiol. 589, 4115-4123 (2011).</li><li>Yang, J., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+P-selectin+glycoprotein+ligand-1:+its+role+as+a+selectin+counterreceptor+in+leukocyte-endothelial+and+leukocyte-platelet+interaction.">The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction.</a> Thromb. Haemost. 81, 1-7 (1999).</li><li>Zarbock, A., Polanowska-Grabowska, R. K., 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=Platelet-neutrophil-interactions:+linking+hemostasis+and+inflammation.">Platelet-neutrophil-interactions: linking hemostasis and inflammation.</a> Blood Rev. 21, 99-111 (2007).</li><li>Chen, J., Lopez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+platelets+with+subendothelium+and+endothelium.">Interactions of platelets with subendothelium and endothelium.</a> Microcirculation. 12, 235-246 (2005).</li><li>Konstantopoulos, 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=Venous+levels+of+shear+support+neutrophil-platelet+adhesion+and+neutrophil+aggregation+in+blood+via+P-selectin+and+beta2-integrin.">Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.</a> Circulation. 98, 873-882 (1998).</li><li>Maugeri, N., de Gaetano, G., Barbanti, M., Donati, M. 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R., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+extracellular+protein+disulfide+isomerase+during+thrombus+formation+in+mice.">A critical role for extracellular protein disulfide isomerase during thrombus formation in mice.</a> J. Clin. Invest. 118, 1123-1131 (2008).</li><li>Cho, 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=Protein+disulfide+isomerase+capture+during+thrombus+formation+in+vivo+depends+on+the+presence+of+beta3+integrins.">Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of beta3 integrins.</a> Blood. 120, 647-655 (2012).</li><li>Gross, P. L., Furie, B. 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The authors declare no competing financial interest.

Here we describe a detailed protocol for real-time fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombosis. Previously, similar fluorescence microscopic approaches were reported to study the molecular mechanism of thrombus formation and vascular inflammation.8,12 Since the heterotypic cell-cell interaction could be important for vaso-occlusion at the injury site, this technology will be a valuabl...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Using a detailed intravital microscopy analysis, heterotypic platelet-neutrophil interactions on the activated endothelium were visualized by infusion of fluorescently-labeled antibodies against a platelet (CD42c) or neutrophil marker (Gr-1) into live mice.
In a model of TNF-&alpha;-induced venular inflammation, most rolling neutrophils were stably adhered to the endothelium presumably by interaction of activated &beta;2 integrins with ICAM-1 during the recording period (3-4.5 hr after injecti...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K ビュー.  University of Illinois at Chicago. ここでは、血管の炎症や生きたマウスにおける血栓形成の間に活性化内皮上の異血小板、好中球の相互作用を可視化する蛍光生体顕微鏡の実験手法を報告する。この顕微鏡技術は、血管疾患の分子メカニズムを研究することが、病態生理学的な条件下での薬理学的薬剤をテストするためにも役立ちます。

ビデオ: Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...