Name: digestion of the murine liver for a flow cytometric analysis of lymphatic endothelial cells
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells at JoVE.com

作者要感谢 gi 和肝脏先天免疫计划为这个项目提供的资金支持。b. a. j. t. 的资金也来自 r01 ai121209。

这里描述的所有方法都已得到科罗拉多大学安舒茨医学校园动物护理和使用机构委员会 (iacuc) 的批准。
1. 材料的制备
<ol><li>在磷酸盐缓冲盐水 (pbs) 中制备 dnase i 的 5 mgml 溶液。</li>
	<li>在点击的 ehaa 培养基中加入 5, 000 u\ l 胶原酶 iv, 制作消化混合物。</li>
	<li>使用前, 将消化混合物在37°c 下加热30分钟。</li>
	<li>在汉克斯平衡盐溶液 (hbss) 中加入4.8% 的...

<ol>
	<li>Tanaka, M., Iwakiri, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatics+in+the+liver.">Lymphatics in the liver.</a> Current Opinion in Immunology. 53, 137-142 (2018).</li><li>Vollmar, B., Wolf, B., Siegmund, S., Katsen, A. D., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+vessel+expansion+and+function+in+the+development+of+hepatic+fibrosis+and+cirrhosis.">Lymph vessel expansion and function in the development of hepatic fibrosis and cirrhosis.</a> The American Journal of Pathology. 151 (1), 169-175 (1997).</li><li>Podgrabinska, 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=Molecular+characterization+of+lymphatic+endothelial+cells.">Molecular characterization of lymphatic endothelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (25), 16069-16074 (2002).</li><li>Cohen, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node-resident+lymphatic+endothelial+cells+mediate+peripheral+tolerance+via+Aire-independent+direct+antigen+presentation.">Lymph node-resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct antigen presentation.</a> Journal of Experimental Medicine. 207 (4), 681-688 (2010).</li><li>Cohen, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerogenic+properties+of+lymphatic+endothelial+cells+are+controlled+by+the+lymph+node+microenvironment.">Tolerogenic properties of lymphatic endothelial cells are controlled by the lymph node microenvironment.</a> PLoS One. 9 (2), e87740 (2014).</li><li>Rouhani, S. 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=Roles+of+lymphatic+endothelial+cells+expressing+peripheral+tissue+antigens+in+CD4+T-cell+tolerance+induction.">Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.</a> Nature Communications. 6, 6771 (2015).</li><li>Tewalt, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+endothelial+cells+induce+tolerance+via+PD-L1+and+lack+of+costimulation+leading+to+high-level+PD-1+expression+on+CD8+T+cells.">Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells.</a> Blood. 120 (24), 4772-4782 (2012).</li><li>Dubrot, 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=Lymph+node+stromal+cells+acquire+peptide-MHCII+complexes+from+dendritic+cells+and+induce+antigen-specific+CD4(+)+T+cell+tolerance.">Lymph node stromal cells acquire peptide-MHCII complexes from dendritic cells and induce antigen-specific CD4(+) T cell tolerance.</a> Journal of Experimental Medicine. 211 (6), 1153-1166 (2014).</li><li>Hirosue, 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=Steady-state+antigen+scavenging,+cross-presentation,+and+CD8++T+cell+priming:+a+new+role+for+lymphatic+endothelial+cells.">Steady-state antigen scavenging, cross-presentation, and CD8+ T cell priming: a new role for lymphatic endothelial cells.</a> Journal of Immunology. 192 (11), 5002-5011 (2014).</li><li>Lund, A. 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=VEGF-C+promotes+immune+tolerance+in+B16+melanomas+and+cross-presentation+of+tumor+antigen+by+lymph+node+lymphatics.">VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics.</a> Cell Reports. 1 (3), 191-199 (2012).</li><li>Lund, A. 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=Lymphatic+vessels+regulate+immune+microenvironments+in+human+and+murine+melanoma.">Lymphatic vessels regulate immune microenvironments in human and murine melanoma.</a> Journal of Clinical Investigation. 126 (9), 3389-3402 (2016).</li><li>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=Immunomodulatory+roles+of+lymphatic+vessels+in+cancer+progression.">Immunomodulatory roles of lymphatic vessels in cancer progression.</a> Cancer Immunology Research. 2 (8), 701-707 (2014).</li><li>Dietrich, T., 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=Cutting+edge:+lymphatic+vessels,+not+blood+vessels,+primarily+mediate+immune+rejections+after+transplantation.">Cutting edge: lymphatic vessels, not blood vessels, primarily mediate immune rejections after transplantation.</a> Journal of Immunology. 184 (2), 535-539 (2010).</li><li>Kedl, 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=Migratory+Dendritic+Cells+acquire+archived+antigen+from+Lymphatic+Endothelial+Cells+for+antigen+presentation+during+lymph+node+contraction.">Migratory Dendritic Cells acquire archived antigen from Lymphatic Endothelial Cells for antigen presentation during lymph node contraction.</a> Nature Communications. 8, 2034 (2017).</li><li>Kedl, R. M., Tamburini, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+archiving+by+lymph+node+stroma:+A+novel+function+for+the+lymphatic+endothelium.">Antigen archiving by lymph node stroma: A novel function for the lymphatic endothelium.</a> European Journal of Immunology. 45 (10), 2721-2729 (2015).</li><li>Tamburini, B. A., Burchill, M. A., Kedl, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+capture+and+archiving+by+lymphatic+endothelial+cells+following+vaccination+or+viral+infection.">Antigen capture and archiving by lymphatic endothelial cells following vaccination or viral infection.</a> Nature Communications. 5, 3989 (2014).</li><li>Yokomori, 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=Lymphatic+marker+podoplanin/D2-40+in+human+advanced+cirrhotic+liver--re-evaluations+of+microlymphatic+abnormalities.">Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver--re-evaluations of microlymphatic abnormalities.</a> BMC Gastroenterology. 10, 131 (2010).</li><li>Nonaka, H., Tanaka, M., Suzuki, K., Miyajima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+murine+hepatic+sinusoidal+endothelial+cells+characterized+by+the+expression+of+hyaluronan+receptors.">Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.</a> Developmental Dynamics. 236 (8), 2258-2267 (2007).</li><li>Dudas, 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=Prospero-related+homeobox+1+(Prox1)+is+a+stable+hepatocyte+marker+during+liver+development,+injury+and+regeneration,+and+is+absent+from+"oval+cells".">Prospero-related homeobox 1 (Prox1) is a stable hepatocyte marker during liver development, injury and regeneration, and is absent from "oval cells".</a> Histochemistry and Cell Biology. 126 (5), 549-562 (2006).</li><li>Schrage, 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=Murine+CD146+is+widely+expressed+on+endothelial+cells+and+is+recognized+by+the+monoclonal+antibody+ME-9F1.">Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1.</a> Histochemistry and Cell Biology. 129 (4), 441-451 (2008).</li><li>Amatschek, 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=Blood+and+lymphatic+endothelial+cell-specific+differentiation+programs+are+stringently+controlled+by+the+tissue+environment.">Blood and lymphatic endothelial cell-specific differentiation programs are stringently controlled by the tissue environment.</a> Blood. 109 (11), 4777-4785 (2007).</li><li>Huang, L., Soldevila, G., Leeker, M., Flavell, R., Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+eliminates+T+cells+undergoing+antigen-triggered+apoptosis+in+vivo.">The liver eliminates T cells undergoing antigen-triggered apoptosis in vivo.</a> Immunity. 1 (9), 741-749 (1994).</li><li>Shay, T., Kang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+Genome+Project+and+systems+immunology.">Immunological Genome Project and systems immunology.</a> Trends in Immunology. 34 (12), 602-609 (2013).</li><li>Li, B., 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=Adult+Mouse+Liver+Contains+Two+Distinct+Populations+of+Cholangiocytes.">Adult Mouse Liver Contains Two Distinct Populations of Cholangiocytes.</a> Stem Cell Reports. 9 (2), 478-489 (2017).</li><li>Randolph, G. J., Ivanov, S., Zinselmeyer, B. H., Scallan, 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=The+Lymphatic+System:+Integral+Roles+in+Immunity.">The Lymphatic System: Integral Roles in Immunity.</a> Annual Review of Immunology. 35, 31-52 (2016).</li><li>Olszewski, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymphatic+system+in+body+homeostasis:+physiological+conditions.">The lymphatic system in body homeostasis: physiological conditions.</a> Lymphatic Research and Biology. 1 (1), 11-21 (2003).</li></ol>

lec 在免疫稳态和调节中的整体重要性最近被曝光25。许多发表的淋巴文献都集中在皮肤和淋巴结上;然而, 淋巴管在全身26被发现 , 因此, 我们需要了解它们在不同器官中的重要性。在这里, 我们展示了一种方法, 其中包括一种逐细胞研究肝脏中的 lec 的方法, 以更好地了解其同时表达的不同表面标记、细胞因子、趋化因子和细胞内蛋白质, 如转录因子。该方法将有?...

淋巴管和 lec 在肝脏中的作用尚不清楚。虽然淋巴血管被发现在肝1的门静脉三位一体和扩大期间的疾病 2, 很少了解在肝脏内的 lec 的功能和表型。随着主要在 lecs3上发现的标记物的发现, 这些细胞在不同组织位中的重要性将在稳态和疾病中填补我们理解中的一个重大空白。lec 通过与 t 细胞4、5、6、7、8、9,10,11,12,13. 淋巴结中的 lec 可通过与迁移的树突状细胞14、15、16的相互作用来促进保护性免疫。因此, 有多个角色的 loec, 这可能是特定的组织和相互作用, 其中存在。然而, 对 lec 如何与组织中的免疫细胞相互作用或 lec 如何在不同器官系统中发挥作用的了解很少;因此, 在肝脏或其他器官内的每个细胞的基础上评估 lec 可能会导致 lec 如何编程组织特异性免疫的进步。虽然许多关注肝脏中 lec 的文献都使用显微镜来可视化 lec, 使用一个或两个标记和形态学 17, 但通过一项研究, 很少用流式细胞仪在细胞上专门评估细胞上的 lec评估了肝窦内皮细胞 (lsecs) 和lec 18之间的差异。通过流式细胞仪分析肝脏中的 lec 群体, 可以深入研究正常稳态或疾病期间的 lec 表型。
为了通过流式细胞仪评估 lec, 需要多个表面标记。通常情况下, lec 是可视化的表达与繁荣相关的同源毒素 1 (prox-1), 淋巴管内皮透明质酸受体 1 (lyve1) 或血管生长因子受体 3 (vegfr3) 使用显微镜。然而, 在肝脏中, 这些标记的表达并不局限于 lec。prox-1 在肝脏发育、再生和损伤19过程中广泛由肝细胞表达, lyve1 和 vegfr3 由肝正弦内皮细胞18表达。在淋巴结中, 利用流式细胞术将 lec 确定为分化 (cd) cd45 (cd31 +)、cd31 + 和 podoplanin + (pdpn)16。然而, 这种方法是太小, 无法分离肝脏中的 lec, 因为 cd45-cd1+ 细胞将捕获内皮细胞, 而肝脏中的血管内皮细胞的主要群体是 lsec。因此, 需要其他标记来区分罕见的 lec 人口和丰富的 lsec 人口。ccux32 (由成熟的 lsecs18表达) 和 cd146 (一种常见的血管内皮细胞标记, 主要由肝脏窦内皮细胞20在肝脏窦内表达, 几乎没有淋巴表达内皮细胞21) 是候选标记。
因此, 我们利用上述标记 cd45、cd31、cd146、cc到146和 pdpn 对肝脏中的 lec 进行了隔离和可视化的方法, 用于流式细胞术。我们描述了使用胶原酶 iv, dnase 1, 和机械分离肝脏组织消化成单细胞悬浮液。我们还描述了使用碘沙诺密度梯度分离非实质细胞 (npc) 和消除细胞碎片。最后, 利用多个标记, 我们确定了以 pdpn 为主要标记的最佳流式细胞术分控策略, 以肝脏中的 lec 为主要标记。

<table>
	<tbody>
		<tr>
			<td>Clicks/EHAA media</td>
			<td>Irvine Scientific</td>
			<td>9195</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Worthington Biochemical corporation</td>
			<td>LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington Biochemical corporation</td>
			<td>LS002145</td>
			<td>Deoxyribonuclease 1</td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>Sigma Aldrich</td>
			<td>D1556</td>
			<td>Density Gradient Medium</td>
		</tr>
		<tr>
			<td>V450 anti mouse CD146(clone ME-9F1</td>
			<td>BD biosciences</td>
			<td>562232</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti mouse CD146 (clone ME-9F1</td>
			<td>Biolegend</td>
			<td>134706</td>
			<td>Fluorescein isothiocyanate (FITC)</td>
		</tr>
		<tr>
			<td>Pacific Blue anti mouse CD31(clone 390)</td>
			<td>Biolegend</td>
			<td>102422</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp/Cy5.5 anti mouse CD31( clone 390)</td>
			<td>Biolegend</td>
			<td>102420</td>
			<td>Peridinin-chlorophyll proteins-Cyanine 5.5 (PerCP-Cy5.5)</td>
		</tr>
		<tr>
			<td>APC anti mouse PDPN (clone 8.1.1)</td>
			<td>Biolegend</td>
			<td>127410</td>
			<td>Allophycocyanin (APC), podoplanin (PDPN)</td>
		</tr>
		<tr>
			<td>APC/Cy7 anti mouse CD45 (clone 30-F11)</td>
			<td>Biolegend</td>
			<td>103116</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti mouse CD45 (clone 30-F11</td>
			<td>Biolegend</td>
			<td>103138</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti mouse CD16/32 (clone 93)</td>
			<td>Biolegend</td>
			<td>101306</td>
			<td>Fluorescein isothiocyanate (FITC)</td>
		</tr>
		<tr>
			<td>PerCp/Cy5.5 anti mouse CD16/32( clone 93)</td>
			<td>Biolegend</td>
			<td>101324</td>
			<td>Peridinin-chlorophyll proteins-Cyanine 5.5 (PerCP-Cy5.5)</td>
		</tr>
		<tr>
			<td>ghost red 780 viability dye</td>
			<td>TONBO biosceinces</td>
			<td>3-0865-T100</td>
			<td></td>
		</tr>
		<tr>
			<td>APC syrian hamster IgG (clone SHG-1)</td>
			<td>Biolegened</td>
			<td>402102</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp/Cy5.5 rat IgG2a (clone RTK2758)</td>
			<td>Biolegend</td>
			<td>400531</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC rat IgG2 (clone eBR2a)</td>
			<td>ebioscience</td>
			<td>1-4321-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti mouse LYVE1 (clone 223322)</td>
			<td>R&#38;D systems</td>
			<td>FAB2125A</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse Cytokeratin(clone EPR17078)</td>
			<td>abcam</td>
			<td>ab181598</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse F4/80 (clone Cl:A3-1)</td>
			<td>Bio-rad</td>
			<td>MCA497</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (fraction V)</td>
			<td>Fischer</td>
			<td>BP1600-100</td>
			<td>Bovine Serum Albumin (BSA)</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>VWR</td>
			<td>E177</td>
			<td>Ethylenediaminetetraacetic acid (EDTA) -for RBC lysis buffer</td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>Fischer</td>
			<td>A687-500</td>
			<td>for RBC Lysis buffer</td>
		</tr>
		<tr>
			<td>Potassium Bicarbonate</td>
			<td>Fischer</td>
			<td>P184-500</td>
			<td>for RBC Lysis buffer</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>2975#21</td>
			<td></td>
		</tr>
		<tr>
			<td>100um cell strainer</td>
			<td>Fischer</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>2.4G2</td>
			<td>in house/ATCC</td>
			<td>ATCC HB-197</td>
			<td>FC block to inhibit non-specific binding to Fc gamma + cells -made from hybridoma</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3788</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical</td>
			<td>Truline</td>
			<td>TR2004</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical</td>
			<td>Falcon</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml Pipete tip</td>
			<td>USA scientific</td>
			<td>1111-2721</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l pipete tip</td>
			<td>USA scientific</td>
			<td>1110-1700</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;l pipete tip</td>
			<td>USA scientific</td>
			<td>1111-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>seriological 10ml pipete</td>
			<td>greiner bio-one</td>
			<td>607107</td>
			<td></td>
		</tr>
		<tr>
			<td>seriological 5ml pipete</td>
			<td>greiner bio-one</td>
			<td>606107</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Fischer</td>
			<td></td>
			<td>Heracell 160i</td>
		</tr>
		<tr>
			<td>BD FacsCanto II flow cytometer</td>
			<td>BD biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clinical Centrifuge</td>
			<td>Beckman coulter</td>
			<td></td>
			<td>model X-14R</td>
		</tr>
	</tbody>
</table>

digestion of the murine liver for a flow cytometric analysis of lymphatic endothelial cells

在肝脏内, 淋巴血管被发现在门静脉三位一体, 其描述的功能是去除间质液体从肝脏到淋巴结, 在那里细胞碎片和抗原可以被调查。我们非常有兴趣了解淋巴血管可能如何参与肝脏内的炎症和免疫细胞功能。然而, 很少有发现建立消化方案, 用于分离淋巴内皮细胞 (lec) 从肝脏或特定的标记, 可用于评估肝脏 lec 的基础上, 每个细胞。因此, 我们优化了肝脏消化和染色的方法, 以评估肝脏中的 lec 群体。我们相信, 这里概述的方法将有助于识别和隔离肝脏中的 lec, 并将加强我们对 lec 如何响应肝脏微环境的理解。

分析肝脏淋巴管的研究主要是使用免疫组织化学来定量肝脏中淋巴管的频率和直径。然而, 这种方法不允许评价 lec 的细胞的基础上, 细胞的基础上, 或表达多个标记, 细胞因子, 趋化因子, 或转录因子。因此, 我们询问是否可以从肝脏中分离肝脏 lec, 并使用流式细胞仪进行评估。以前的工作分离淋巴结 lec 是使用自由酶 dl (胶原酶-i------------------------<...

Watch this Scientific Journal Video about Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells at JoVE.com

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

该方案的目标是使用所述标记识别肝脏内的淋巴管内皮细胞群。我们利用胶原酶 iv 和 dnase 和组织的温和切碎, 结合流式细胞仪, 以确定一个独特的淋巴内皮细胞的群体。

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 ...

此方法可以帮助回答淋巴和肝脏领域的关键问题，如淋巴内皮细胞或 LECs 如何响应特定刺激，以及这如何有助于疾病发病机制。该技术的主要优点是，我们可以评估肝淋巴内皮细胞表型和功能基于每个细胞的基础上，并可用于细胞排序后下游应用。虽然这种方法可以提供肝脏淋巴生物学的见解，它也可以用来评估不同组织的淋巴内皮细胞，如皮肤和乳腺。演示这个程序的将是杰弗里·芬隆，一个专业的研究助理，从我的实验室。要准备从分离的肝细胞的单细胞悬浮液，首先用70%的乙醇喷洒给小鼠，并固定爪子到解剖板。使用解剖剪刀切割离动物的约一厘米的皮肤，并使用齿形钳将皮肤从身体中抬离。在皮肤和内膜之间插入剪刀尖，打开剪刀分离组织，然后继续皮肤切口到颈部。将皮肤固定到每个前肢下和每个后肢上方的解剖板上，向上拉包头袋，将切口伸向颈部。抓住肝脏的叶，切在胸骨的下方，让肝脏周围解剖。然后将器官转移到四毫升的点击的EHAA介质。使用手术刀将肝脏切成直径约一毫米的碎片，并消化在五百微升新鲜准备的消化液和五百微升的DNase 1。在三十七摄氏度下十五分钟后，使用五百毫升移液器将组织彻底混合，将容器再返回孵化器十五分钟。在孵育结束时，通过一百微米过滤器将消化的样品转移到一个五十毫升的圆锥管中，然后使用一毫升注射器中的柱塞，通过网状体轻轻按压剩余的组织。用五毫升隔离缓冲液清洗过滤器，然后轻轻按压过滤器中剩余的组织。通过离心收集分离的肝细胞，并在四毫升红血球解液缓冲液中重新悬浮颗粒。在室温下五分钟后，将细胞用十毫升的隔离缓冲液清洗，并计算血细胞仪上的细胞，以确定完整的肝脏计数。将颗粒重新悬浮在五毫升20%碘糖醇中，用一毫升PBS覆盖细胞悬浮液。通过密度梯度分离分离细胞，收获PBS和碘桑醇之间的层。然后通过一百微米滤株过滤细胞，进入一个新的五十毫升锥形管。将细胞用10毫升的新鲜分离缓冲液清洗，并在PBS的500微升中重新悬浮产生的颗粒，并辅以2%的胎儿牛血清或FBS。计数后，等分约五倍十至第六非乳质细胞进入每个对照和实验井的96井板离心和重新悬浮在PBS加FBS每井90微升的颗粒。将适当的实验和控制原抗体鸡尾酒添加到每口油井中，并用适当的生存能力标记染色所有油井。在这个程序中最重要的步骤是抗体的正确滴定和使用冰型控制和荧光-1控制，以验证染色。在摄氏四度下30分钟后，离心机用每井100微升PBS加FBS清洗细胞，将每个井的细胞转移到单独的5毫升圆底流细胞学管中，达到每管PBS加FBS的四百微升最终体积。然后，使用少量细胞调整流式细胞仪上的激光和补偿设置，并读取每个管的所有事件。读取所有管后，将数据导入适当的流式细胞仪分析程序。基于负表达的活体标记染料的活细胞上的栅极。基于细胞的大小和粒度及其生存能力标记染料表达的活细胞上的门，然后使用适当的等型控制和荧光-1数据对CD45阴性CD31阳性细胞群进行浇注，以确定阳性和负量。然后根据CD16/32和足二丁酮表达对CD45阴性CD31阳性细胞进行门控，以识别CD45阴性CD31阳性、CD16/32阴性足腺素阳性淋巴内皮，以及CD45阴性CD31阳性、CD16/32阳性足阴性肝胆内皮细胞群。强调内皮细胞表达淋巴血管和内皮催眠一，但不是CD146。肝脏分离CD31阳性CD16/32阳性细胞分离，如证明也是CD146阳性和足科素阳性，而CD31阳性CD16/32阴性细胞主要是CD146阴性细胞，以及足科素阳性或阴性。根据荧光-1染色确定积极性。肝脏淋巴内皮细胞为CD146阴性，PDPN阳性。血管内皮和穆林肝巨噬细胞都不表达足骨素。波多普兰宁染色也可用于区分胆管细胞和淋巴细胞体基于胆管的不同核结构以及细胞蛋白7染色。由于肝脏中只有淋巴内皮细胞共同表达血管内皮生长因子受体3和普罗斯佩罗家血箱蛋白1，这些标记可用于进一步确认肝脏淋巴内皮细胞种群的纯度。在尝试此过程时，重要的是要记住快速工作并保持细胞在冰上。按照此过程，可以执行其他方法，如 LEC 群体的流动排序，以回答其他问题，如 LEC 的转录分析。这项技术开发后，为肝脏特异性淋巴生物学领域的研究人员探索淋巴如何影响小鼠和人类肝脏疾病铺平了道路。一般来说，新方法的个体将挣扎，因为淋巴内皮细胞是肝脏中不常见、渐进的群体，需要快速进行。

Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

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

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

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