Name: isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting
Uploaded: 2015-05-01T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting at JoVE.com

लेखकों बेकर फाउंडेशन और Zerina Lokmic के समर्थन रॉयल बच्चों के फाउंडेशन &#39;विज्ञान फैलोशिप में महिलाओं को स्वीकार करना होगा। एंड्रयू जी Elefanty और एडुअर्ड जी स्टेनली NHMRC वरिष्ठ रिसर्च फैलो हैं। उनके प्रयोगशालाओं में काम NHMRC और स्टेम सेल ऑस्ट्रेलिया द्वारा समर्थित किया गया।

आचार बयान: लसीका कुरूपता और चमड़ी के ऊतकों के संग्रह के लिए नैतिक अनुमोदन रॉयल बच्चों के अस्पताल, मेलबोर्न, ऑस्ट्रेलिया में मानव अनुसंधान आचार समितियों से प्राप्त हुई थी। हस्ताक्षर किए गए सहमति सर्जर...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+microcirculation.+Tissue+engineering.">Engineering the microcirculation. Tissue engineering.</a> Part B, Reviews. 14 (1), 87-103 (2008).</li><li>Lokmic, Z., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+and+stereological+assessment+of+blood+and+lymphatic+vessels.">Visualisation and stereological assessment of blood and lymphatic vessels.</a> Histol Histopathol. 26 (6), 781-796 (2011).</li><li>Hagerling, 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=A+novel+multistep+mechanism+for+initial+lymphangiogenesis+in+mouse+embryos+based+on+ultramicroscopy.">A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy.</a> EMBO J. 32 (5), 629-644 (2013).</li><li>Pollmann, C., Hagerling, R., Kiefer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+lymphatic+vessel+development,+growth,+and+function.">Visualization of lymphatic vessel development, growth, and function.</a> Adv Anat Embryol Cell Biol. 214, 167-186 (2014).</li><li>Breiteneder-Geleff, 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=Angiosarcomas+express+mixed+endothelial+phenotypes+of+blood+and+lymphatic+capillaries:+podoplanin+as+a+specific+marker+for+lymphatic+endothelium.">Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.</a> Am J Pathol. 154 (2), 385-394 (1999).</li><li>Kaipainen, 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=Expression+of+the+fms-like+tyrosine+kinase+4+gene+becomes+restricted+to+lymphatic+endothelium+during+development.">Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.</a> Proc Natl Acad Sci U S A. 92 (8), 3566-3570 (1995).</li><li>Schacht, V., 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=T1alpha/podoplanin+deficiency+disrupts+normal+lymphatic+vasculature+formation+and+causes+lymphedema.">T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.</a> EMBO J. 22 (14), 3546-3556 (2003).</li><li>Kriehuber, 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=Isolation+and+characterization+of+dermal+lymphatic+and+blood+endothelial+cells+reveal+stable+and+functionally+specialized+cell+lineages.">Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.</a> J Exp Med. 194 (6), 797-808 (2001).</li><li>Florez-Vargas, 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=Comparative+analysis+of+D2-40+and+LYVE-1+immunostaiing+in+lymphatic+malformations.">Comparative analysis of D2-40 and LYVE-1 immunostaiing in lymphatic malformations.</a> Lymphology. 41 (3), 103-110 (2008).</li><li>Makinen, 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=PDZ+interaction+site+in+ephrinB2+is+required+for+the+remodeling+of+lymphatic+vasculature.">PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.</a> Genes Dev. 19 (3), 397-410 (2005).</li><li>Connell, F. 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LECs द्रव समस्थिति, विदेशी एंटीजन और अवशोषण और कुछ पोषक तत्वों के परिवहन के लिए प्रतिरक्षा प्रतिक्रिया को बनाए रखने में एक महत्वपूर्ण भूमिका निभाते हैं। LEC समस्थिति ऐसे जीवाणु संक्रमण और ट्यूमर मेटास्ट?...

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies (Gibco)</td>
			<td>11965-092</td>
			<td>Used to collect tissue samples from the operating theatre.</td>
		</tr>
		<tr>
			<td>EGM-2 MV Bullet Kit</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>EGM-2 MV Bullet Kit contains 500 ml of EBM-2 media and human EGF, hydrocortisone, gentamycin (GA-1000), fetal bovine serum (FBS), VEGF, human FGF-b, R3-IGF-1 and ascorbic acid.</td>
		</tr>
		<tr>
			<td>VEGF-C</td>
			<td>R&#38;D Systems</td>
			<td>2179-VC-025</td>
			<td>Complete endothelial cell media is supplemented with 50 ng/mL VEGF-C</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution (100x)</td>
			<td>Life Technologies (Gibco)</td>
			<td>15240-062</td>
			<td>This solution contains 10,000U/ml of penicillin, 10,000 mg/ml of streptomycin and 25 mg/ml of Fungizone antimycotic. Use at 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Used at 10 mg/ml concentrations. It can be re-used for up to 1 month if kept at 4 degrees and maintained sterile. Use 7 mL to coat 150 cm2 flask.</td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell dissociation reagent</td>
			<td>Life Technologies (Gibco)</td>
			<td>A11105-01</td>
			<td>StemPro&#174;Accutase&#174; is used at 0.05 ml per cm2 to cover the entire surface area of the flask. Using lesser volumes may result in incomplete cell detachment.</td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue dye</td>
			<td>Life Technologies (Gibco)</td>
			<td>15250-061</td>
			<td>Used as a viability stain.</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche Applied Biosciences</td>
			<td>4942078001</td>
			<td>Used at 0.04%</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Worthington Lab</td>
			<td>4176</td>
			<td>Used at 0.25%</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche Applied Biosciences</td>
			<td>11284932001</td>
			<td>Used at 0.01%</td>
		</tr>
		<tr>
			<td>70 mm nylon cell strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated VEGFR-3 clone 9D9F9, clone WM59</td>
			<td>BioLegend</td>
			<td>356204</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated CD34, clone 581</td>
			<td>BioLegend</td>
			<td>343516</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti human CD31, clone WM59</td>
			<td>BioLegend</td>
			<td>303116,</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated rat anti human PODOPLANIN, clone NC-80.</td>
			<td>BioLegend</td>
			<td>337006</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>PE-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400112</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400126</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400120</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated mouse IgG2a, k isotype, clone RATK2758</td>
			<td>BioLegend</td>
			<td>400525</td>
			<td>Used at 1:200 dilution</td>
		</tr>
	</tbody>
</table>

isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting

ऐसे प्राथमिक lymphedema, लसीका विकृतियों और लसीका ट्यूमर के रूप में लसीका प्रणाली विकारों महत्वपूर्ण रुग्णता का कारण है, लेकिन थोड़ा उनके जीव विज्ञान के बारे में जाना जाता है कि दुर्लभ स्थितियों रहे हैं। रोगग्रस्त और स्वस्थ ऊतकों से अत्यधिक शुद्ध मानव लसीका endothelial कोशिकाओं (LECs) अलग, आनुवंशिक आणविक और सेलुलर स्तर पर लसीका अन्तःचूचुक के अध्ययन की सुविधा होगी। यह इन जांच के लिए इन शर्तों के नैदानिक ​​प्रबंधन परिवर्तित हो सकता है कि नए उपचार के लिए लक्ष्य प्रकट हो सकता है कि अनुमान है। LECs और लसीका कुरूपता लसीका endothelial कोशिकाओं (एल एम LECs) चमड़ी मानव के अलगाव का वर्णन एक प्रोटोकॉल प्रस्तुत किया है। प्राप्त करने के लिए एक एकल कक्ष निलंबन ऊतक कीमा और enzymatically dispase द्वितीय और collagenase द्वितीय का उपयोग कर इलाज किया गया था। जिसके परिणामस्वरूप एकल कक्ष निलंबन फिर भेदभाव (सीडी) मार्कर CD34, CD31, संवहनी endothelial वृद्धि कारक-3 (VEGFR-3) और PODOPLANIN के क्लस्टर के लिए एंटीबॉडी के साथ चिह्नित किया गया। Staiनेड व्यवहार्य कोशिकाओं अन्य endothelial और गैर endothelial कोशिकाओं से CD34 कम CD31 स्थिति VEGFR -3 स्थिति PODOPLANIN स्थिति एलएम LEC आबादी को अलग करने के लिए एक fluorescently सक्रिय सेल सॉर्टर (FACS) पर हल किया गया। छाँटे गए एलएम LECs सुसंस्कृत थे और आगे प्रयोगात्मक उपयोग के लिए फ़ाइब्रोनेक्टिन-लेपित बोतल पर विस्तार किया।

प्रारंभिक ऊतक पाचन के बाद, unfractionated नमूनों की संस्कृति में 24 घंटे के बाद, अलग endothelial सेल कालोनियों एक साथ fibroblast कोशिकाओं की तरह है और चिकनी मांसपेशियों की कोशिकाओं के साथ (2A चित्रा) मनाया जा सकता है। बाद छं...

Watch this Scientific Journal Video about Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting at JoVE.com

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

इस प्रोटोकॉल के लक्ष्य प्रतिदीप्ति सक्रिय सेल छँटाई (FACS) का उपयोग मानव लसीका कुरूपता पुटी-तरह के जहाजों और foreskins अस्तर लसीका endothelial कोशिकाओं को अलग करने के लिए है। इसके बाद सेल संवर्धन और इन कोशिकाओं के विस्तार, आनुवंशिक प्रोटिओमिक, कार्यात्मक और सेल भेदभाव के अध्ययन के लिए प्रयोगात्मक मिलावट के एक नए स्तर परमिट।

मल्टी पैरामीटर प्रतिदीप्ति सक्रिय सेल छँटाई द्वारा मानव लिम्फेटिक endothelial कोशिकाओं का अलगाव

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

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Medicine

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

कैंसर कोशिकाओं द्वारा Endothelial सेल monolayer के आक्रमण उपाय करने के लिए एक वास्तविक समय विद्युत प्रतिबाधा आधारित तकनीक

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

मानव दंत लुगदी स्टेम सेल के अलगाव विशेषता, और तुलनात्मक भेदभाव दो अलग अलग तरीकों का उपयोग करके स्थायी दांत से व्युत्पन्न

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

असामान्य सूत्रीविभाजन साथ एसोसिएटेड सेल डेथ लाइव कैंसर कोशिकाओं में confocal इमेजिंग द्वारा मनाया

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

मानव भ्रूण अग्नाशय सेल समूहों के अलगाव, संस्कृति, और इमेजिंग

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

मानव प्रोस्टेट कैंसर के नमूनों से कैंसर स्टेम कोशिकाओं के अलगाव

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

अग्नाशय के कैंसर नई उपचार रणनीतियों के विकास के लिए सेल के लक्षण स्टेम का अध्ययन

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

ट्यूमर विशिष्ट endothelial कोशिकाओं के अलगाव और संस्कृति के विस्तार

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

अलगाव और कुत्ते धमनियों और नसों से प्राथमिक endothelial कोशिकाओं की संस्कृति

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

स्वस्थ स्वयंसेवकों और उनके प्रवासी संभावित सीरम नमूनों से प्रभावित कार्डिएक सर्जरी के बाद से endothelial पूर्वज कोशिकाओं का अलगाव

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

तैयार करना और<em&gt; इन विट्रो</em&gt; चुम्बकीय की विशेषता मीर संशोधित अंतर्कलीय कोशिकाओं

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...