Name: blocking lymph flow by suturing afferent lymphatic vessels in mice
Uploaded: 2020-05-14T14:00:00
Description: Watch this Scientific Journal Video about Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice at JoVE.com

作者感谢艾娃·扎迪内扎德对手稿进行校对。这项工作得到了加拿大健康研究所（CIHR，PJT-156035）和加拿大SL创新基金会（32930）以及中国国家自然科学基金会（81901576）的支持。

所有动物工作都需要得到机构和政府伦理及动物处理委员会的批准。 这是一个非生存手术。
1. 材料准备
<ol><li>通过将 70 mL 的 100% 乙醇与 30 mL 的无菌水混合，准备 100 mL 的 70% 乙醇。手术前将所有手术工具绝育，并在手术前和手术期间将工具保持为 70% 乙醇，以保持灭菌。</li>
	<li>准备注射装置。<ol>
<li>切割 ±30 厘米聚乙烯管（直径 0.28 毫米）。将 30 G x 1/2 针（针 A）的尖端?...

<ol>
	<li>Pflicke, H., Sixt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preformed+portals+facilitate+dendritic+cell+entry+into+afferent+lymphatic+vessels.">Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels.</a> The Journal of Experimental Medicine. 206, 2925-2935 (2009).</li><li>Schmid-Schonbein, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microlymphatics+and+lymph+flow.">Microlymphatics and lymph flow.</a> Physiological Reviews. 70, 987-1028 (1990).</li><li>Skalak, T. C., Schmid-Schonbein, G. W., Zweifach, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+morphological+evidence+for+a+mechanism+of+lymph+formation+in+skeletal+muscle.">New morphological evidence for a mechanism of lymph formation in skeletal muscle.</a> Microvascular Research. 28, 95-112 (1984).</li><li>Johnston, M. G., Hay, J. B., Movat, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+prostaglandin+production+in+various+inflammatory+lesions,+measured+in+draining+lymph.">Kinetics of prostaglandin production in various inflammatory lesions, measured in draining lymph.</a> The American Journal of Pathology. 95, 225-238 (1979).</li><li>Eisenhoffer, J., Yuan, Z. Y., Johnston, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+L-arginine+pathway+plays+a+role+in+the+regulation+of+pumping+activity+in+bovine+mesenteric+lymphatic+vessels.">Evidence that the L-arginine pathway plays a role in the regulation of pumping activity in bovine mesenteric lymphatic vessels.</a> Microvascular Research. 50, 249-259 (1995).</li><li>Gasheva, O. Y., Zawieja, D. C., Gashev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraction-initiated+NO-dependent+lymphatic+relaxation:+a+self-regulatory+mechanism+in+rat+thoracic+duct.">Contraction-initiated NO-dependent lymphatic relaxation: a self-regulatory mechanism in rat thoracic duct.</a> Journal of Physiology. 575, 821-832 (2006).</li><li>Breslin, J. W., 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=Vascular+endothelial+growth+factor-C+stimulates+the+lymphatic+pump+by+a+VEGF+receptor-3-dependent+mechanism.">Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism.</a> American Journal of Physiology- Heart and Circulatory Physiology. 293, 709-718 (2007).</li><li>Randolph, G. J., Angeli, V., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic-cell+trafficking+to+lymph+nodes+through+lymphatic+vessels.">Dendritic-cell trafficking to lymph nodes through lymphatic vessels.</a> Nature Reviews. Immunology. 5, 617-628 (2005).</li><li>Mempel, T. R., Henrickson, S. E., Von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases.">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427, 154-159 (2004).</li><li>Gerner, M. Y., Casey, K. A., Kastenmuller, W., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+and+antigen+dispersal+landscapes+regulate+T+cell+immunity.">Dendritic cell and antigen dispersal landscapes regulate T cell immunity.</a> The Journal of Experimental Medicine. 214, 3105-3122 (2017).</li><li>Kastenmuller, W., Torabi-Parizi, P., Subramanian, N., Lammermann, T., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatially-organized+multicellular+innate+immune+response+in+lymph+nodes+limits+systemic+pathogen+spread.">A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread.</a> Cell. 150, 1235-1248 (2012).</li><li>Gerner, M. Y., Torabi-Parizi, P., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategically+localized+dendritic+cells+promote+rapid+T+cell+responses+to+lymph-borne+particulate+antigens.">Strategically localized dendritic cells promote rapid T cell responses to lymph-borne particulate antigens.</a> Immunity. 42, 172-185 (2015).</li><li>Moussion, C., Girard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+control+lymphocyte+entry+to+lymph+nodes+through+high+endothelial+venules.">Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules.</a> Nature. 479, 542-546 (2011).</li><li>Gretz, J. E., Norbury, C. C., Anderson, A. O., Proudfoot, A. E., Shaw, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph-borne+chemokines+and+other+low+molecular+weight+molecules+reach+high+endothelial+venules+via+specialized+conduits+while+a+functional+barrier+limits+access+to+the+lymphocyte+microenvironments+in+lymph+node+cortex.">Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex.</a> The Journal of Experimental Medicine. 192, 1425-1440 (2000).</li><li>Mebius, R. E., Breve, J., Duijvestijn, A. M., Kraal, 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+function+of+high+endothelial+venules+in+mouse+lymph+nodes+stimulated+by+oxazolone.">The function of high endothelial venules in mouse lymph nodes stimulated by oxazolone.</a> Immunology. 71, 423-427 (1990).</li><li>Mebius, R. E., Streeter, P. R., Breve, J., Duijvestijn, A. M., Kraal, 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+influence+of+afferent+lymphatic+vessel+interruption+on+vascular+addressin+expression.">The influence of afferent lymphatic vessel interruption on vascular addressin expression.</a> Journal of Cell Biology. 115, 85-95 (1991).</li><li>Mebius, R. E., 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=Expression+of+GlyCAM-1,+an+endothelial+ligand+for+L-selectin,+is+affected+by+afferent+lymphatic+flow.">Expression of GlyCAM-1, an endothelial ligand for L-selectin, is affected by afferent lymphatic flow.</a> Journal of Immunology. 151, 6769-6776 (1993).</li><li>Drayton, D. L., Liao, S., Mounzer, R. H., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+organ+development:+from+ontogeny+to+neogenesis.">Lymphoid organ development: from ontogeny to neogenesis.</a> Nature Immunology. 7, 344-353 (2006).</li><li>Tomei, A. A., Siegert, S., Britschgi, M. R., Luther, S. A., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+flow+regulates+stromal+cell+organization+and+CCL21+expression+in+a+tissue-engineered+lymph+node+microenvironment.">Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment.</a> Journal of Immunology. 183, 4273-4283 (2009).</li><li>Liao, S., Jones, D., Cheng, G., Padera, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+quantitative+measurement+of+collecting+lymphatic+vessel+contraction+in+mice.">Method for the quantitative measurement of collecting lymphatic vessel contraction in mice.</a> Journal of Biological Methods. 1, 6 (2014).</li><li>Lin, 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=Perinodal+Adipose+Tissue+Participates+in+Immune+Protection+through+a+Lymphatic+Vessel-Independent+Route.">Perinodal Adipose Tissue Participates in Immune Protection through a Lymphatic Vessel-Independent Route.</a> Journal of Immunology. 201, 296-305 (2018).</li><li>Gardenier, J. 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=Diphtheria+toxin-mediated+ablation+of+lymphatic+endothelial+cells+results+in+progressive+lymphedema.">Diphtheria toxin-mediated ablation of lymphatic endothelial cells results in progressive lymphedema.</a> Journal of Clinical Investigation Insight. 1, 84095 (2016).</li></ol>

在健康和患病条件下，阻断淋巴流动在操纵抗原输送到LN方面有着广泛的应用。可以使用此方法控制抗原传递的时间，以研究连续淋巴流如何调节排空 LNs 中的免疫反应。这种淋巴流中断方法还可用于研究淋巴如何影响 LN 中的细胞分块、细胞激活、细胞迁移和细胞细胞相互作用。
专门表达人类白喉毒素受体（DTR）的小鼠在淋巴内皮细胞（Flt4-cre-dtr）中已经开发出来;这?...

淋巴系统的空间组织提供结构和功能支持，以有效地去除细胞外液体，将抗原和抗原呈现细胞（APC）运送到排出的LNs。最初的淋巴血管（也称为淋巴毛细血管）由于其不连续的细胞间结，便于从周围的细胞外空间1有效地收集液体、细胞和其他材料， 因此具有高度渗透性。最初的淋巴血管合并成收集淋巴血管，这些血管有紧密的细胞间结，连续的地下室膜和淋巴肌肉覆盖。收集淋巴血管负责运输收集的淋巴到排水的LNs，并最终返回淋巴循环2，3。将淋巴推进到排水的LN的收集淋巴血管是发性淋巴血管4，5，6，7。淋巴血管的阻塞可以阻止淋巴流进入LNs，这是研究淋巴流动功能时有用的技术。
先前的研究表明，淋巴流动在运输抗原和药水，以及维持LN平衡方面起着重要作用。众所周知，组织衍生的APCs，通常激活的迁移树突状细胞（DCs），通过发源淋巴血管前往LN激活T细胞8。自由形式的抗原，如微生物或可溶性抗原，被动地与淋巴流动到LN激活LN居民APC的想法在过去十年中得到了接受9，10，11，12。与淋巴一起携带的自由形式抗原在感染后需要几分钟时间才能到达 LN，LN-驻留细胞活化可能在刺激后 20 分钟内发生。这比迁移 DC 的激活要快得多，迁移 DC 进入排空 LN 9 需要 8小时多。除了运输抗原以启动免疫保护外，淋巴还携带细胞因子和DC到LN，以维持其微环境，并支持免疫细胞平衡13，14。此前，通过阻断淋巴血管来阻断淋巴流动表明，淋巴需要维持支持恒流T细胞所需的HV表型，B细胞向LN15、16、17定位。CCL21是一种关键的化学素，它指导直流和T细胞在LN8，18中的定位。阻塞淋巴流会中断 LN 中的 CCL21 表达，并可能中断 LN19中的直流和 T 细胞定位和/或相互作用。因此，阻断淋巴流动可以通过破坏调节 LN 免疫反应的 LN 微环境，直接或间接地消除抗原/直流对排干 LN 的访问。为了更好地研究淋巴流动的功能，提出了一个实验方案（图1），通过将支脚的淋巴血管从脚垫到pLN的吸血，来阻止小鼠的淋巴流动。该方法是今后在健康和患病条件下研究淋巴功能的重要技术。

<table>
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride Saline</td>
			<td>Baxter</td>
			<td>JB1323</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Greenfield Global</td>
			<td></td>
			<td>University of Calgary distribution services UN1170.</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Nair Sensitive Formula Hair Removal Cr&#232;me with Sweet Almond Oil and Baby Oil, 200-ml. Or similar product.</td>
		</tr>
		<tr>
			<td>Evans Blue dye</td>
			<td>Sigma Life Science</td>
			<td>E2129-10G</td>
			<td>For 1 ml of Evans blue dye, add 0.1g Evans blue to 10 ml PBS. The Evens Blue solution will be filtered through 0.22 mm filters and kept sterile in 1ml aliquots.</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I (FITC)</td>
			<td>Sigma Life Science</td>
			<td>F7250-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #3</td>
			<td>WPI</td>
			<td>500337</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5</td>
			<td>WPI</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection apparatus</td>
			<td></td>
			<td></td>
			<td>Connect one end of polyethylene tubing to 30G &#215; &#189; needle. Attach a 1ml TB syringe to the needle. Dislodge needle shaft from another 30G &#215; &#189; needle. Insert the blunt end of the 30G &#215; &#189; needle shaft into the other end of the tubing. The inside diameter of this tubing is 0.28mm. Thus, 1.6 cm of fluid in the tubing is 1 &#956;l.</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>IRIS Forcep straight</td>
			<td>WPI</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>IRIS scissors</td>
			<td>WPI</td>
			<td>14218-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Narketan</td>
			<td>DIN 02374994</td>
			<td>The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.</td>
		</tr>
		<tr>
			<td>Needles (26Gx3/8)</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>305110</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (30Gx1/2)</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Paton Needle Holder</td>
			<td>ROBOZ</td>
			<td>RS6403</td>
			<td>Straight, Without Lock; Serrated</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma Life Science</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape (1.25cmx9.1m )</td>
			<td>Transpore</td>
			<td>1527-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape (2.5cmx9.1m )</td>
			<td>Transpore</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Davis and Geck CYANAMID Canada</td>
			<td>11/04</td>
			<td>0.7 metric monofilament polypropylene</td>
		</tr>
		<tr>
			<td>Syringe (1ml)</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>VANNAS scissors</td>
			<td>World Precision Instruments (WPI)</td>
			<td>14122-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Rompun</td>
			<td>DIN02169606</td>
			<td>The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Olympus S261 (522-STS OH141791) with light source: Olympus Highlight 3100</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td>SP8</td>
		</tr>
	</tbody>
</table>

blocking lymph flow by suturing afferent lymphatic vessels in mice

淋巴血管通过运输抗原、细胞因子和细胞排干淋巴结（LNs）来保持组织流动性平衡和优化免疫保护至关重要。淋巴流中断是研究淋巴血管功能的重要方法。从杂物足部到流行淋巴结（pLNs）的淋巴血管被明确界定为淋巴排入pLNs的唯一途径。给这些发性淋巴血管进行吸管可以选择性地防止淋巴流向pLAN。此方法允许干扰淋巴流，对排水 pLN 中的淋巴内皮细胞、支淋巴血管以及该区域周围的其他淋巴血管的损害最小。此方法已用于研究淋巴如何影响 LN 中的高内皮淋巴瘤（ HEV ）和化疗表达，以及淋巴如何在没有功能性淋巴血管的情况经 LN 周围的脂肪组织。随着人们越来越认识到淋巴功能的重要性，这种方法将有更广泛的应用，以进一步解开淋巴血管在调节LN微环境和免疫反应的功能。

淋巴细胞缝合在以前的研究中已经使用15，16，17，19，作为一个重要的工具，研究淋巴流的功能之前，淋巴细胞的分子生物学被更好地理解。阻塞淋巴流中断LN平衡，这导致 HEV 失去最佳淋巴细胞宿主到 LN15、16、 17所需的关键基因表达。<sup...

Watch this Scientific Journal Video about Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice at JoVE.com

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

提出了一种通过手术阻断淋巴血管来阻止淋巴流动的方案。

通过在小鼠中吸液性淋巴血管阻止淋巴流

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

此方法以最小的淋巴内皮细胞损伤中断淋巴流动，并精确控制阻断时间，可用于研究淋巴流如何影响淋巴结中的平衡和免疫反应。帮助演示这个程序的将是我实验室的研究生薛静娜。要准备注射装置，切割约30厘米的聚乙烯管。将针尖 A 连接到管的一端。小心地将另一根针断开，将断裂的一侧连接到聚乙烯管的另一端。然后将针头 A 连接到一毫升的结核素注射器上。使用前，立即在盐水中准备10：1氯胺酮-锡氨酸混合物。麻醉小鼠后，注射250微升的氯胺酮/木胺混合物内，确保完全麻醉与头趾捏。用剪发器在腿部周围剃毛，然后在腿部周围涂抹脱毛霜。五分钟后，用湿润的组织擦去残留的毛皮和脱毛霜，然后用无菌水清洁腿部。在腿部周围喷洒 70%乙醇，对操作区域进行消毒。将鼠标放在易发位置，并使用手术胶带将手术区域露出右腿。在贸易中，将5微升的1%埃文斯蓝色染料，或9厘米的液体从注射装置管到鼠标的右脚垫。轻轻按摩足垫，帮助液体进入淋巴血管。在解剖显微镜下，从波莱特叶的下边缘定位一个切口部位五毫米。用一把剪刀，做一个大约五毫米长的小切口。然后使用精细的操作钳来拉伸切口并暴露收集的淋巴血管。识别导致流行淋巴结的两个发性淋巴血管。使用针架，小心地将缝合针插入支点淋巴血管和下垂动脉之间。轻轻地将针头拉出，围绕发状淋巴血管。小心地拉缝合线，留下大约两厘米的缝合线。使用针架帮助打结。轻轻按摩脚垫，以确保没有埃文斯蓝色染料通过缝合现场，然后用剪刀切割多余的字符串。缝合其他发性淋巴血管，然后用用于淋巴血管的相同缝合关闭皮肤切口。对于假控制，在左脚垫中注入5微升1%的埃文斯蓝色染料，并按摩脚垫。用切口打开皮肤，然后闭合伤口，而不使血管破裂。当监测小鼠手术后，右腿应显示水肿，与埃文斯蓝色染料蔓延到大腿，而控制腿将显示埃文斯蓝色染料限制在脚垫。手术后，在右脚垫和左侧立即注射10微升2%FITC。FITC注射后2、6和12小时，从安乐死小鼠的fossa收集流行淋巴结。小心地去除 pLNs 周围的佩里诺达尔脂肪组织。将 pLN 嵌入最佳切割温度化合物中，将模干窦区域朝向低温模具的一侧。使用低温准备 20 微米冷冻部分。要确定 pLN 中的 FITC 分布，请用共和显微镜对低温部分进行成像。在 FITC 注射后两、六和 12 小时拍摄的共和图像显示，在使发淋巴血管苏化后，FITC 在流行淋巴结中的积累大大减少。pLNS 中的残留 FITC 优先积聚在淋巴结鼻窦中。近脂肪组织的共生图像显示，当淋巴血管被苏化阻塞时，FITC进入组织时淋巴结鼻窦，但它没有有效地分布在整个淋巴结。尝试此方法时，重要的是要记住，在血管和淋巴血管之间插入缝合针至关重要。此步骤的故障可能会使船只中断。执行此方法后，有可能为感染或其他免疫刺激的小鼠接种疫苗，以研究淋巴流如何调节免疫保护。淋巴流的功能不被很好地理解。此方法可用于研究淋巴结中没有淋巴流动的细胞迁移。

Research

JoVE Journal

Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

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

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

在肠系膜淋巴管空心​​大鼠模型：应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

细胞内染色和流式细胞仪识别鼠主动脉，肾脏和淋巴结内淋巴细胞亚群的高血压模型

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

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

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

高频超声监测小鼠瘀血致深静脉血栓形成

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...