This work was supported by the German Ministry for Education and Research (BMBF 01EO1003 and BMBF 01EO1503).

Experiments were conducted on male mice age 8 to 12 weeks old under the approval of the ethics committee on animal experimentation from Rhineland-Palatinate (authorization number 23 177-07/G12-1-002 and 23 177-07/G15-1-051).
1. Mouse Anesthesia and Surgery Preparation
<ol>
	<li>Prior to surgery, verify the good health and condition of the animals. Before surgery, monitor animals once per day for 2-3 days for their general condition (appearance, posture, spontaneous behavior) as well as body weight, food, and water consumption.</li>
	<li>Prepare the master mix for anesthesia with midazolam (5 mg/kg body weight), medetomidine (0.5 mg/kg body weight), and fentanyl (0.05 mg/kg body weight) to intraperitoneally (i.p.) inject 200 &#181;L/30 g mouse in 1 mL syringe with a 26 G needle.</li>
	<li>Osmotic pump implantation and animal preparation for IVM experiments are performed on an operation field with a 39 &#176;C warming platform. Sterilize all tools in a disinfection bath and disinfect the warming platform.</li>
	<li>During the procedures, protect the animal eyes with eye ointment. Remove the fur with razors before osmotic pump implantation. Use a hair removal cream and cotton swabs for isolation of carotids and jugular catheter implantation in IVM experiments. Disinfect the animal skin using two skin disinfectants: one antiseptic and disinfectant containing povidone iodine and one skin antiseptic containing octenidine in alcohol-based solution.&#160;</li>
	<li>Prepare the master mix to antagonize anesthesia (&#34;antisedan mix&#34;) with atipamezol (0.05 mg/kg body weight) and flumazenil (0.01 mg/kgbody weight), and prepare 200 &#181;L/mouse in 1 mL syringe with a 26 G needle to be subcutaneously (s.c.) injected.</li>
</ol>
2. Osmotic Pump Preparation and Implantation
NOTE: The subcutaneous AngII infusion using osmotic pumps was described in detail by Lu et al.17
<ol>
	<li>Prepare the AngII by reconstituting the lyophilized powder with sterile saline and adjust the concentration with the animal weight and the delivery rate of the pump. Keep the reconstituted AngII in plastic tubes (do not use glass tubes).</li>
	<li>Fill the pumps with the AngII solution to deliver 1 mg/(kg&#183;d) for 7 days. After the preparation, keep the pumps in sterile saline for 4-6 h minimum at 37 &#176;C.</li>
	<li>Test the complete anesthesia of the mouse after injection of the anesthesia mix with rear foot reflexes and place it on the warm operation field. Anesthesia induction takes 10-15 min and works better if animals are placed in a dark environment.</li>
	<li>Shave the lower back of the mouse and disinfect it with an alcoholic skin antiseptic.</li>
	<li>Make a 1 cm incision perpendicular to the spine using scissors. Create a pocket for the pump using a strait hemostat and insert the pump (flow moderator first). The pocket must allow free movement of the pump with no pressure on the wound.</li>
	<li>Close the wound with sutures, and not clips, in order to limit inflammation; then disinfect with skin antiseptic.</li>
	<li>Antagonize the anesthesia by s.c. injection of 200 &#181;L of the antisedan mix and return the mouse to its cage.</li>
	<li>Carry out postoperative analgesia with buprenorphine (0.075 mg/kg s.c.) one time after the surgery and on demand depending on the animal behavior.</li>
</ol>
3. Jugular Catheter Implantation and Carotids Preparation
<ol>
	<li>Test the complete anesthesia of the mouse after injection of the anesthesia mix with rear food reflexes and place the anesthetized animal in dorsal recumbence on the 39 &#176;C surgical plate&#160;with a rectal probe in order to maintain the temperature and put the ointment over the eyes to protect them during the procedure.</li>
	<li>Remove neck hairs using the hair removal cream. Use a cotton swab to spread and rub the cream for 2 min until the hairs start to fall out. After an additional 2 min, remove the cream and hairs with a spatula and disinfect the skin with an alcoholic skin antiseptic.</li>
	<li>Perform the surgery under a stereomicroscope. Make a 1-1.5 cm long incision in the skin longitudinally in the neck, 1 cm next to the trachea. Then make two other incisions perpendicularly to both extremities of the first incisions.</li>
	<li>Carefully remove tissues next to the skin and remove the piece of skin covering the left jugular vein and the trachea.</li>
	<li>Carefully isolate the parotid gland; sublingual and submaxillary glands from the zone of interest with 2 curved forceps. Do not cut or damage the glands, place them to allow the best access to the jugular vein and to the carotids.</li>
	<li>To isolate the jugular vein, use thin forceps and slowly open and close it to gently free the vessel from the surrounding tissue. Once the vein is completely clean, position the forceps under the vessel and place two 7-0 sutures of 10 cm each under it.</li>
	<li>Close the suture proximal to the head with two knots. On the other suture, prepare one knot but do not close it.</li>
	<li>Hold the catheter (0.28 mm inside diameter, 0.61 mm outer diameter) filled with 37 &#176;C sterile saline with forceps with one hand; while with the other hand, make a small incision into the vessel with a thin scissors or a curved 26 G needle. Then insert the catheter into the jugular vein and close the knot two times. Close the suture proximal to the head over the catheter in order to ensure complete immobilization.</li>
	<li>To isolate and prepare the carotid arteries, dissect the area covering the trachea using thin curved forceps.</li>
	<li>Once the carotids are free from surrounding tissues, remove the vagus nerve (it looks like a white line adjacent to the carotid) from the vessel (but do not cut it). The thin forceps can be closed and slowly opened to gently mobilize the nerve.</li>
	<li>Once both arteries are completely clean, place the forceps under the first carotid artery and place a small black 3 mm wide x 2.5 cm length plastic piece under the vessel. It is very important not to overstretch the vessel and to check that the blood flow is present once the vessel holder is placed. Place the second artery over the vessel holder. If the mouse is not placed directly under the microscope, put back the glands over the trachea and apply 37 &#176;C sterile saline to avoid drying of the zone.</li>
</ol>
4. Assessment of Rolling and Adhering Leukocytes/LysM+ Cells with IVM
<ol>
	<li>Prepare acridine orange from a 2 mg/mL stock solution stored at -20 &#176;C and dilute it 1:4 in sterile saline (0.5 mg/mL final concentration) in a 1 mL syringe. Protect the syringe from light and use 200 &#181;L per one mouse.
	<ol>
		<li>Test different conditions: (1) Injection of acridine orange with the jugular catheter; (2) Injection of acridine orange directly into the tail lateral vein (with this condition no jugular catheter was implanted); (3) Measurement without acridine orange using the fluorescence LysMCre+IRG+ mice (here again no jugular catheter was implanted).</li>
	</ol>
	</li>
	<li>Once the catheter is in place and the two carotids are isolated, position the mouse under the microscope. Perform measurements with a high-speed wide-field fluorescence microscope using a long-distance condenser and a 10X (NA 0.3) water immersion objective with a monochromator, a beam splitter, and a charge-coupled device camera. Perform image acquisition and analysis with real-time imaging system.</li>
	<li>Focus the microscope objective in the middle of the carotid artery at the endothelium surface. Slowly inject 50 &#181;L of acridine orange (0.5 mg/mL) (take into account the dead volume of the catheter for the first injection). Set the software to record 100 images with an exposure time of 120 milliseconds per image.</li>
	<li>Make four videos per carotid artery (left and right) at different locations on the artery for each video. If the fluorescence signal decreases, inject another 50 &#181;L of acridine orange.</li>
	<li>After recording all the videos euthanize the animal by cervical dislocation.</li>
	<li>Set the video speed at 10 images per second and quantify the rolling and adherent cells in a 200 &#181;m x 250 &#181;m rectangle view placed in the middle of the vessel for each video. Count rolling and adherent cells; adherent cells are defined as cells that do not move or detach from the endothelium within the 10 s video. To simplify quantification, remove the channel with the red fluorescence and use only the green fluorescence to count rolling and adhering cells.</li>
	<li>For experiments using acridine orange, make a manual threshold of the fluorescence in order to remove background made by endothelial cells.</li>
</ol>

<ol>
	<li>Lewington, S., 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=Age-specific+relevance+of+usual+blood+pressure+to+vascular+mortality:+a+meta-analysis+of+individual+data+for+one+million+adults+in+61+prospective+studies.">Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies.</a> Lancet. 360, 1903-1913 (2002).</li><li>Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mosaic+theory+revisited:+common+molecular+mechanisms+coordinating+diverse+organ+and+cellular+events+in+hypertension.">The mosaic theory revisited: common molecular mechanisms coordinating diverse organ and cellular events in hypertension.</a> J Am Soc Hypertens. 7, 68-74 (2013).</li><li>Guzik, T. 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=Role+of+the+T+cell+in+the+genesis+of+angiotensin+II+induced+hypertension+and+vascular+dysfunction.">Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction.</a> J Exp Med. 204, 2449-2460 (2007).</li><li>De Ciuceis, C., 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=Reduced+vascular+remodeling,+endothelial+dysfunction,+and+oxidative+stress+in+resistance+arteries+of+angiotensin+II-infused+macrophage+colony-stimulating+factor-deficient+mice:+evidence+for+a+role+in+inflammation+in+angiotensin-induced+vascular+injury.">Reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin II-infused macrophage colony-stimulating factor-deficient mice: evidence for a role in inflammation in angiotensin-induced vascular injury.</a> Arterioscler Thromb Vasc Biol. 25, 2106-2113 (2005).</li><li>Wenzel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme+M-positive+monocytes+mediate+angiotensin+II-induced+arterial+hypertension+and+vascular+dysfunction.">Lysozyme M-positive monocytes mediate angiotensin II-induced arterial hypertension and vascular dysfunction.</a> Circulation. 124, 1370-1381 (2011).</li><li>Kossmann, S., 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=Platelet-localized+FXI+promotes+a+vascular+coagulation-inflammatory+circuit+in+arterial+hypertension.">Platelet-localized FXI promotes a vascular coagulation-inflammatory circuit in arterial hypertension.</a> Sci Transl Med. 9, (2017).</li><li>Rodriguez-Iturbe, B., Pons, H., Johnson, R. J. <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+the+Immune+System+in+Hypertension.">Role of the Immune System in Hypertension.</a> Physiol Rev. 97, 1127-1164 (2017).</li><li>Masedunskas, A., 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=Intravital+microscopy:+a+practical+guide+on+imaging+intracellular+structures+in+live+animals.">Intravital microscopy: a practical guide on imaging intracellular structures in live animals.</a> Bioarchitecture. 2, 143-157 (2012).</li><li>Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy:+a+novel+tool+to+study+cell+biology+in+living+animals.">Intravital microscopy: a novel tool to study cell biology in living animals.</a> Histochem Cell Biol. 133, 481-491 (2010).</li><li>Wenzel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+oxygenase-1+suppresses+a+pro-inflammatory+phenotype+in+monocytes+and+determines+endothelial+function+and+arterial+hypertension+in+mice+and+humans.">Heme oxygenase-1 suppresses a pro-inflammatory phenotype in monocytes and determines endothelial function and arterial hypertension in mice and humans.</a> Eur Heart J. 36, 3437-3446 (2015).</li><li>Pawelski, H., Lang, D., Reuter, S. <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+monocytes+and+platelets:+implication+for+life.">Interactions of monocytes and platelets: implication for life.</a> Front Biosci (Schol Ed). 6, 75-91 (2014).</li><li>Hinsbergh, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium--role+in+regulation+of+coagulation+and+inflammation.">Endothelium--role in regulation of coagulation and inflammation.</a> Semin Immunopathol. 34, 93-106 (2012).</li><li>Njock, M. S., 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=Endothelial+cells+suppress+monocyte+activation+through+secretion+of+extracellular+vesicles+containing+antiinflammatory+microRNAs.">Endothelial cells suppress monocyte activation through secretion of extracellular vesicles containing antiinflammatory microRNAs.</a> Blood. 125, 3202-3212 (2015).</li><li>Colognato, R., 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=Differential+expression+and+regulation+of+protease-activated+receptors+in+human+peripheral+monocytes+and+monocyte-derived+antigen-presenting+cells.">Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells.</a> Blood. 102, 2645-2652 (2003).</li><li>Schaffner, A., Rhyn, P., Schoedon, G., Schaer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+expression+of+platelet+factor+4+in+human+monocytes--role+of+PARs+as+a+quantitatively+important+monocyte+activation+pathway.">Regulated expression of platelet factor 4 in human monocytes--role of PARs as a quantitatively important monocyte activation pathway.</a> J Leukoc Biol. 78, 202-209 (2005).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+Angiotensin+II+Infusion+using+Osmotic+Pumps+Induces+Aortic+Aneurysms+in+Mice.">Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice.</a> J Vis Exp. , (2015).</li><li>Koh, T. J., DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+wound+healing:+the+role+of+the+macrophage.">Inflammation and wound healing: the role of the macrophage.</a> Expert Rev Mol Med. 13, e23 (2011).</li><li>Dargaud, Y., 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=Proposal+for+standardized+preanalytical+and+analytical+conditions+for+measuring+thrombin+generation+in+hemophilia:+communication+from+the+SSC+of+the+ISTH.">Proposal for standardized preanalytical and analytical conditions for measuring thrombin generation in hemophilia: communication from the SSC of the ISTH.</a> J Thromb Haemost. , (2017).</li></ol>

The authors have nothing to disclose

LysM+ monocytes were previously shown to be implicated in the development of hypertension5. Here we show that LysM+ immune cells roll and adhere to the endothelial layer in response to AngII infusion. This finding was obtained using LysMCre+IRG+ mice from our previous studies exploring the role of immune cells in hypertension in vivo by visualizing leukocytes with injection of acridine orange6,10. Thus, discrimination of cell types adhering to the endothelium was not possible.
The IVM is a very useful tool for vascular studies and in vivo observations of cells directly in the vasculature, but the impact of injecting dyes with the help of a surgically inserted catheter in the inflammatory context of hypertension remains unknown. To evaluate the potential effect of catheter and acridine orange injection we took advantage of the LysMCre+IRG+ mice. AngII infusion increased the number of rolling LysM+ cells to the endothelium. Insertion of a catheter into the jugular vein amplified the effect and more rolling and adhering leukocytes were detected in AngII-infused mice instrumented with a catheter compared to mice without catheter implants. This indicates that implantation of a carotid catheter causes a systemic inflammatory reaction in the context of wound healing that overlays with the immune reaction seen in hypertension18. In addition, due to the proximity of the jugular vein to the carotid artery an additional immune activation might have occurred by affecting the jugular vein. Since this effect was not present when injections were made directly into the tail vein, we can assume that the effect was due not to the dye but to the procedure of inserting the catheter. We demonstrated here the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.
One critical step of the procedure is the AngII infusion. Tail cuff assessment of the blood pressure can be made in order to control the effectiveness of AngII delivery by the pump. Blood pressure should increase after 2&#8722;3 days of infusion. To limit inflammation that could influence LysM+ cells activation, closure of the incision where the pump is implanted should be made with sutures instead of clips. One limitation must be noted about tail vein injection: to make the best possible data acquisition, 4 videos are usually taken for each carotid. If the fluorescent signal decreases, 50 &#181;L of acridine orange are injected but with tail injection it is more difficult to make several injections and to keep the carotids stable under the microscope objective. The duration of the presence of a catheter in the jugular vein might also modulate the immune cells activation since it is affecting the coagulation activation in platelet-rich plasma prepared 4 h after the catheter implantation19. This aspect of catheter implantation needs further investigation.
Finally, even if we appreciate the impact of jugular catheter implantation on LysM+ cell activation after AngII infusion, we cannot fully estimate the role of the preparation, skin removal, and carotid isolation on a potential LysM+ cell activation. We conclude that the use of fluorescence reporter mice like the LysMCre+IRG+ mouse is recommended to avoid disruption of vessel integrity during the animal preparation for in vivo imaging.

Arterial hypertension increases the risk of cardiovascular disease and death and promotes the development of atherosclerosis, coronary heart disease, and arterial or venous thromboembolisms1. The development of hypertension depends on the interaction of environmental, genetic, endocrine, and hemodynamic factors. Currently, immunity and related inflammation are appreciated to play an important role in the etiology of hypertension2.
Among immune cells, T lymphocytes as well as monocytes and macrophages were found to be causally involved in AngII-induced vascular inflammation and hypertension, in part related to their ability to trigger reactive oxygen species3. Macrophage colony-stimulating factor deficient mice showed reduced response to AngII regarding blood pressure increase and vascular inflammation4. In a previous work, we could show that LysM+ monocytes drive vascular dysfunction and inflammation in AngII-induced hypertension5. More recently, we described a novel pathway in which coagulation factor XI cooperates with platelets and the vessel wall to induce thrombin-dependent vascular inflammation6. The current knowledge about the role of the immune system in hypertension was recently summarized and reviewed by Rodriguez-Iturbe et al.7
Since the involvement of immune cells in the development of hypertension became evident, models and techniques to study the interaction between the vessel and immune cells became necessary. Epifluorescence IVM of blood vessels is a useful tool to observe in vivo interactions between circulating blood cells and the endothelium8,9,10. With this technique, injection of dyes intercalating with DNA (such as acridine orange) can visualize nucleated cells (circulating as well as from the endothelium). Isolated platelets stained ex vivo with rhodamin-6G or dichlorofluorescein (DCF) can be injected to visualize platelet-rich thrombus in arterial or venous injury models.
Typically, a jugular vein catheter is used to inject tracers or marked platelets. Alteration of the endothelium and subsequent activation of the coagulation cascade are both known to have effects on monocyte activation. Endothelial injury immediately leads to platelet activation via subendothelial matrix molecules to seal the tissue, with ensuing monocyte attraction and activation11. On the contrary, an intact endothelium is known to have anticoagulant properties (e.g., via tissue factor pathway inhibitor or thrombomodulin)12 and direct inhibitory effects on the monocyte, e.g., through the secretion of extracellular vesicles containing microRNAs13. Monocytes are known to produce a tissue factor, the extrinsic activator of the coagulation cascade and express protease-activated receptors (PARs) that can be activated by thrombin and participate in monocyte activation14,15. Therefore, any activation of platelets or of the coagulation cascade due to vascular injury may have unexpected effects on monocyte activation and interfere with the observed phenomenon. With the help of IRG transgenic LysM Cre transgenic mice, a double-fluorescent Cre reporter mouse (LysMCre+IRG+), we propose to study in detail the effect of injections with a catheter and alternative methods on LysM+ myelomonocytic cells in a mouse model of arterial hypertension16.

<table>
	<tbody>
		<tr>
			<td>Midazolam</td>
			<td>Ratiopharm GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Pfizer Deutschland GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Janssen-Cilag GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Alzane</td>
			<td>Zoetis</td>
			<td></td>
			<td>Antisedan mix</td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Hikma pharma</td>
			<td></td>
			<td>Antisedan mix</td>
		</tr>
		<tr>
			<td>Braunol</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Octeniderm</td>
			<td>Sch&#252;lke</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic pumps</td>
			<td>Alzet</td>
			<td>1007D</td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>Angiotensin II</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>7-0 prolene suture</td>
			<td>Ethicon</td>
			<td></td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>Acridine orange</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Smiths Medical Deutschland GmbH</td>
			<td></td>
			<td>Inside diameter, 0.28 mm; outer diameter, 0.61 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI fluorescence microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam Splitter</td>
			<td>Photometrics</td>
			<td>CMR-DV2-SYS - DualView2 MicroImager</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UMPLFLN10X</td>
			<td>Water immersion objective with a monochromator</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>ORCA-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and analysis software</td>
			<td>Olympus</td>
			<td>Realtime Imaging System eXcellence RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>Jax 008705</td>
			<td>B6.Cg-Tg(CAG-DsRed,-EGFP)5Gae/J</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>Jax 004781</td>
			<td>LysM Cre (B6.129P2-Lyz2tm1(cre)Ifo/J )</td>
		</tr>
	</tbody>
</table>

visualizing leukocyte rolling and adhesion in angiotensin ii-infused mice: techniques and pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

Carotids of LysMCre+IRG+ mice infused with AngII were observed using IVM. Acridine orange was injected using a jugular catheter. We aimed to look at the proportion of LysM+ cells in contact with the vascular wall compared to all nucleated circulating cells (which could also interact with the vessel since discrimination of the cell type interacting with the vessel remained an open question from our previous work). At baseline, the presence of a jugular catheter causes adhesion of LysM+ cells (Figure 2A, D). After acridine orange injection, the same cells were fluorescent, but also the endothelial cells were fluorescent (Figure 2B, E). After the reduction of the background to limit endothelial related fluorescence, the data indicate that all nucleated adhering cells are LysM+ (Figure 2C, F); these results hold true after AngII infusion confirming our previous results and demonstrating that the LysMCre+IRG+ is a good model to observe the effect of AngII on LysM+ cell activation.
We evaluated the role of administration routes of acridine orange on LysM+ cell activation after AngII infusion in LysMCre+IRG+. After one week of AngII infusion, leukocyte endothelium interactions in carotid arteries of LysMCre+IRG+ mice were imaged and visualized by IVM with or without acridine orange injection via a jugular catheter or through the tail vein. Without any catheter or injection, rolling of LysM+ cells was significantly increased after AngII infusion compared to untreated mice, and adhesion showed an increase (Figure 3). Injection of acridine orange with a jugular catheter increased adhesion and rolling in AngII treated mice to a greater extent compared with mice without a catheter (Figure 3). Injection of acridine orange in the tail vein leads to similar adhesion and rolling compared to the mice that did not receive injection of acridine orange (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56948/56948fig1.jpg" /> 
Figure 1. Scheme for angiotensin II infusion and assessment of LysM+ cells rolling and adhesion in mice. LysMCre+IRG+ mice were infused 7 days with AngII and adhering as well as rolling LysM+ cells over the carotids were quantified with or without injection of acridine orange through a jugular catheter or by tail vein injection. <a href="https://cloudflare.jove.com/files/ftp_upload/56948/56948fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56948/56948fig2.jpg" /> 
Figure 2. Assessment of LysM+ cell adhesion and rolling in carotid arteries of LysMCre+IRG+ mice. Adhesion of LysM+ cells in LysMCre+IRG+ mice before (A, D) and after (B, E) acridine injection via a jugular catheter. Red and green fluorescence were recorded, LysM+ cells (in green) and smooth muscle cells (in red) were visible. After injection of acridine orange, endothelial cells are also visible in green but removing background fluorescence allows only circulating nucleated cell visualization (C, F). <a href="https://cloudflare.jove.com/files/ftp_upload/56948/56948fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56948/56948fig3.jpg" /> 
Figure 3. Evaluation of administration routes of fluorescent dyes in angiotensin II infused LysMCre+IRG+ mice. Quantification of rolling (A) and adhering (B) LysM+ cells in sham operated or AngII-infused animals and with or without injection of acridine orange using a jugular catheter or by tail vein injection. Representative pictures of the different conditions (C). Results are mean &#177; standard error of the mean. 2-way ANOVA was performed and Bonferroni&#39;s post hoc test, n = 3-12/groups; *p &#60; 0.05. <a href="https://cloudflare.jove.com/files/ftp_upload/56948/56948fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls at JoVE.com

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

visualizing-leukocyte-rolling-adhesion-angiotensin-ii-infused-mice

Research

JoVE Journal

Immunology and Infection

8.6K Views.  University Medical Center Mainz. This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium. 

Video: Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

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 ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

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. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

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

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 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

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 ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...