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

The authors would like to thank the GI and Liver Innate Immune Programs for monetary support of this project. B.A.J.T. is also funded by R01 AI121209.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus.
1.&#160;Preparation of the Materials
<ol>
	<li>Make a 5 mg/mL solution of DNase I in phosphate-buffered saline (PBS).</li>
	<li>Make a digestion mixture by adding 5,000 U/mL of collagenase IV to Click&#8217;s EHAA media.</li>
	<li>Warm the digestion mixture at 37 &#176;C for 30 min prior to use.</li>
...

<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>

The overall importance of LECs in immune homeostasis and regulation has recently come to light25. Much of the published lymphatic literature focuses on skin and lymph nodes; however, lymphatics are found throughout the body26 and, thus, our understanding of their importance in different organs is needed. Here we show a method in which LECs in the liver can be studied on a cell-by-cell basis to better understand their concurrent expression of different surface markers, cytok...

The role of lymphatic vessels and LECs in the liver is not well understood. While lymphatic vessels are found within the portal triad of the liver1 and expand during disease2, very little is understood regarding the function and phenotype of LECs within the liver. With the discovery of markers that are found primarily on LECs3, the importance of these cells within different tissue niches in homeostasis and disease will fill a significant gap in our understanding. LECs have a major role in maintaining peripheral tolerance in the lymph node and in metastatic tumors by interacting directly with T cells4,5,6,7,8,9,10,11,12,13. LECs in the lymph node can promote protective immunity via their interactions with migratory dendritic cells14,15,16. Therefore, there are multiple roles for LECs which may be specific to the tissues and interactions in which they are present. However, very little is understood about how LECs interact with immune cells in the tissue or how LECs function in different organ systems; thus, evaluating LECs on a per cell basis within the liver or other organs may lead to advances in how LECs program tissue-specific immunity. While much of the literature that focuses on LECs in the liver uses microscopy to visualize LECs using one or two markers and morphology17, very little has been done to specifically evaluate LECs on a cell by cell basis using flow cytometry, though one study did evaluate differences between liver sinusoidal endothelial cells (LSECs) and LECs18. Being able to analyze LEC populations in the liver by flow cytometry allows for the in-depth study of LEC phenotype during normal homeostasis or disease.
To evaluate LECs by flow cytometry, multiple surface markers are needed. Typically, LECs are visualized by the expression of prospero-related homeobox 1 (Prox-1), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) or vascular endothelial growth factor receptor 3 (VEGFR3) using microscopy. However, in the liver, the expression of these markers is not restricted to LECs. Prox-1 is widely expressed by hepatocytes during liver development, regeneration, and injury19, and LYVE1 and VEGFR3 are expressed by the liver sinusoidal endothelial cells18. In the lymph node, LECs are identified using flow cytometry as clusters of differentiation (CD) CD45-, CD31+, and podoplanin+ (PDPN)16. However, this approach is too minimal to isolate LECs in the liver since CD45- CD31+ cells will capture endothelial cells, and the predominant population of vascular endothelial cells in the liver are LSECs. Thus, other markers are needed to distinguish the rare LEC population from the abundant LSEC population. Both CD16/32 (expressed by mature LSECs18) and CD146 (a common vascular endothelial cell marker that is predominately expressed within the liver sinusoids by liver sinusoidal endothelial cells20 with little to no expression by lymphatic endothelial cells21) were candidate markers.
Therefore, we optimized a method for isolating and visualizing LECs in the liver using the above markers, CD45, CD31, CD146, CD16/32, and PDPN for flow cytometry. We describe the use of collagenase IV, DNase 1, and mechanical separation for liver tissue digestion into a single-cell suspension. We also describe the use of iodixanol density gradient for the isolation of non-parenchymal cells (NPC) and to eliminate cellular debris. Finally, using multiple markers, we determine the optimal flow cytometry gating strategy to identify LECs from the liver with PDPN as the predominant marker.

<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

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 lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

Studies analyzing liver lymphatics have primarily used immunohistochemistry to quantitate the frequency and diameter of lymphatic vessels in the liver. However, this method does not allow for the evaluation of LECs on a cell-by-cell basis or for expression of multiple markers, cytokines, chemokines, or transcription factors. Therefore, we asked whether liver LECs could be isolated from the liver and evaluated using flow cytometry. Previous work isolating lymph node LECs was performed usin...

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

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

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 ...

This method can help answer key questions in lymphatic and liver fields, such as how lymphatic endothelial cells, or LECs, respond to specific stimuli and how that contributes to disease pathogenesis. The main advantage of this technique is that we can evaluate liver lymphatic endothelial cell phenotype and function on a per cell basis, and can be used for down-stream applications after cell sorting. Though this method can provide insight into liver lymphatic biology, it can also be used to evaluate lymphatic endothelial cells from different tissues, such as skin and mammary gland.Demonstrating the procedure will be Jeffrey Finlon, a professional research assistant from my laboratory. To prepare a single-cell suspension from isolated liver cells, begin by spraying down the mouse with seventy-percent ethanol, and securing the paws to a dissection board. Use dissection scissors to cut the skin about one centimeter above the anus and use toothed forceps to lift the skin away from the body.Insert the scissor tips between the skin and the peritoneum and open the scissors to separate the tissues before continuing the skin incision up to the neck. Secure the skin to the dissection board under each forelimb and above each hindlimb, and pull the peritoneal sack upward to extend the incision toward the neck. Grasp the lobes of the liver and cut just below the sternum to allow dissection around the liver.Then transfer the organ into four milliliters of Click's EHAA medium. Use the scalpel to cut the liver into approximately one-millimeter diameter pieces and digest the pieces in five hundred microliters of freshly prepared digestion solution and five hundred microliters of DNase 1. After fifteen minutes at thirty-seven degrees Celsius, use a five hundred milliliter pipet to mix the tissues thoroughly and return the container to the incubator for another fifteen minutes.At the end of the incubation, transfer the digested sample through a one hundred micron strainer into a fifty milliliter conical tube and use the plunger from a one milliliter syringe to gently press the remaining pieces of tissue through the mesh. Wash the filter with five milliliters of isolation buffer and gently press any remaining tissue through the strainer. Collect the isolated liver cells by centrifugation and re-suspend the pellet in four milliliters of red blood cell lysis buffer.After five minutes at room temperature, wash the cells in ten milliliters of isolation buffer and count the cells on the hemocytometer to determine the full liver count. Re-suspend the pellet in five milliliters of twenty-percent iodixanol and overlay the cell suspension with one milliliter of PBS. Separate the cells by density gradient separation and harvest the layer between the PBS and the iodixanol.Then filter the cells through a one hundred micrometer strainer into a new fifty milliliter conical tube. Wash the cells in ten milliliters of fresh isolation buffer and re-suspend the resulting pellet in five hundred microliters of PBS, supplemented with two-percent fetal bovine serum, or FBS. After counting, aliquot approximately five times ten to the sixth non-parenchymal cells into each control and experimental well of a ninety-six well plate for centrifugation and re-suspend the pellets in ninety microliters of PBS plus FBS per well.Add the appropriate experimental and control primary antibody cocktails to each well and stain all of the wells with an appropriate viability marker. The most important step in this procedure is the proper titration of the antibodies and the use of ice type controls and fluorescence-1 controls to validate the staining. After thirty minutes at four degrees Celsius, centrifuge wash the cells with one hundred microliters of PBS plus FBS per well and transfer the cells from each well into individual five milliliter round-bottom flow cytometry tubes to a four hundred microliter final volume of PBS plus FBS per tube.Then, use a small volume of cells to adjust the laser and compensation settings on the flow cytometer and read all of the events for each tube. When all of the tubes have been read, import the data into an appropriate flow cytometry analysis program. Gate on live cells based on the viability marker dye where negative expression is considered live.Gate on the live cells based on the size and granularity of the cells as well as their viability marker dye expression, followed by gating on the CD45 negative CD31 positive cell population using the appropriate isotype control and fluorescence-1 data to determine the positive and negative populations. Then gate the CD45 negative CD31 positive cells according to their CD16/32 and podoplanin expression to allow identification of the CD45 negative CD31 positive, CD16/32 negative podoplanin positive lymphatic endothelial, and CD45 negative CD31 positive, CD16/32 positive podoplanin negative liver sinusoidal endothelial cell populations. Emphatic endothelial cells express lymphatic vessel and endothelial hyaluronan one, but not CD146.Liver isolated CD31 positive CD16/32 positive cells isolated as demonstrated are also CD146 positive and podoplanin positive, while the CD31 positive CD16/32 negative cells are primarily CD146 negative and either podoplanin positive or negative. Positivity was determined based on fluorescence-1 staining. The liver lymphatic endothelial cells are CD146 negative and PDPN positive.Neither the vascular endothelium nor murine liver macrophages express podoplanin. Podoplanin staining can also be used to distinguish cholangiocytes from lymphatic vessels based on the distinct nuclear structures of the bile ducts as well as by cytokeratin 7 staining. As only lymphatic endothelial cells co-express vascular endothelial growth factor receptor 3 and Prospero homeobox protein 1 in the liver, these markers can be further used to confirm the purity of the sorted liver lymphatic endothelial cell population.While attempting this procedure, it is important to remember to work quickly and keep cells on ice. Following this procedure, other methods like flow sorting of LEC populations can be performed in order to answer additional questions, such as transcriptional profiling of LECs. After its development, this technique paved the way for researchers in the field of liver-specific lymphatic biology to explore how lymphatics impact liver disease in mice and humans.Generally, individuals new to this method will struggle because lymphatic endothelial cells are an infrequent, gradual population in a liver and the method needs to be performed quickly.

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Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

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

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

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

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

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

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

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

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

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

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

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 microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

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 Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

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

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

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

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

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