Name: seven steps to stellate cells
Uploaded: 2011-05-10T13:00:00
Description: Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

इस काम बायोमेडिकल अनुसंधान और NIH AI083426 01-RO1 (परिवार कल्याण) में उत्कृष्टता के लिए स्मिथ परिवार पुरस्कार द्वारा वित्त पोषित किया गया था. पैट्रिक Maschmeyer और मेलनी Flach Boehringer Ingelheim फाउंडेशन से पीएचडी फैलोशिप द्वारा समर्थित हैं.

C57BL 6 / चूहों ~ उम्र के 20 सप्ताह या पुराने पर किया जाना चाहिए. पुरुष 25-30g वजन चूहों के इस्तेमाल की सिफारिश की है. तारामय कोशिकाओं की उपज चूहों को 2 महीने के लिए एक विटामिन एक समृद्ध आहार खिलाने से पहले सेल अलगाव ?...

<ol>
	<li>Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

यकृत तारामय कोशिकाओं को जिगर में आवश्यक शारीरिक और pathophysiological प्रक्रियाओं को विनियमित. इसके अलावा, तारामय कोशिकाओं प्रतिजन पेश गुणों के अधिकारी, उन्हें यकृत प्रतिरक्षा प्रतिक्रिया का एक महत्वपूर्ण घटक ...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> कैटलॉग सं. </th></tr><tr><td> 70μm सेल छलनी </td><td> BD </td><td> 352350 </td></tr><tr><td> CaCL 2 x 2H 2 हे </td><td> सिग्मा Aldrich </td><td> C3306 </td></tr><tr><td> Collagenase पी </td><td> रॉश </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11960 </td></tr><tr><td> DNase मैं </td><td> रॉश </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21-031-cv </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> भ्रूण गोजातीय सीरम </td><td> Gibco </td><td> 16000 </td></tr><tr><td> ग्लूकोज़ </td><td> सिग्मा Aldrich </td><td> G7528 </td></tr><tr><td> ढाल centrifugation ट्यूबों </td><td> Greiner जैव - एक </td><td> 163160 </td></tr><tr><td> HEPES </td><td> Gibco </td><td> 15630 </td></tr><tr><td> KCL </td><td> सिग्मा Aldrich </td><td> P9541 </td></tr><tr><td> 2 के.एच. पीओ 4 </td><td> सिग्मा Aldrich </td><td> P9791 </td></tr><tr><td> एल-Glutamine </td><td> Gibco </td><td> 25030 </td></tr><tr><td> MgCl 2 x 6 2 हे </td><td> Fluka </td><td> 63068 </td></tr><tr><td> MgSO 4 x 7 2 हे </td><td> सिग्मा Aldrich </td><td> M2773 </td></tr><tr><td> ना दो चार HPO 2H एक्स 2 हे </td><td> Fluka </td><td> 71643 </td></tr><tr><td> NaCl </td><td> सिग्मा Aldrich </td><td> S3014 </td></tr><tr><td> नाह 2 4 एक्स एच 2 हे पीओ </td><td> Fluka </td><td> 71507 </td></tr><tr><td> 3 NaHCO </td><td> Fluka </td><td> 71628 </td></tr><tr><td> Nycodenz </td><td> Accudenz एजी </td><td> AN7050/BLK </td></tr><tr><td> पेनिसिलीन / स्ट्रेप्टोमाइसिन </td><td> Gibco </td><td> 15140 </td></tr><tr><td> क्रमिक वृत्तों में सिकुड़नेवाला पम्प </td><td> कोल Parmer </td><td> HV-7523-70 </td></tr><tr><td> Phenol लाल </td><td> सिग्मा Aldrich </td><td> P4633 </td></tr><tr><td> Pronase ई </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> सिलिकॉन ट्यूब </td><td> Masterflex </td><td> एचवी 96,440 - 14 </td></tr><tr><td> सोडियम पाइरूवेट </td><td> Gibco </td><td> 11360 </td></tr><tr><td> टिशू कल्चर फ्लास्क (2 25cm) </td><td> BD </td><td> 353108 </td></tr><tr><td> पंख आसव सेट </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

यहाँ हम माउस जिगर से यकृत तारामय कोशिकाओं के अलगाव के लिए एक विधि का वर्णन. तारामय सेल शुद्धि के लिए, माउस यकृत को पचा रहे हैं<em&gt; बगल में</em&gt; और<em&gt; इन विट्रो में</em&gt; Pronase - collagenase घनत्व ढाल centrifugation के लिए पहले उपचार के द्वारा. इस तकनीक अत्यधिक शुद्ध यकृत तारामय कोशिकाओं पैदावार.

सात कदम कक्ष तारामय

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Research

JoVE Journal

Immunology and Infection

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomatal रिस्पांस मूल्यांकन लाइव बैक्टीरियल कोशिकाओं पूरी पत्ती इमेजिंग का उपयोग

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

सेल भाग्य का पालन<em&gt; ई. कोलाई</em&gt; फेज Lambda द्वारा संक्रमण के बाद

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

एक संशोधित EPA के 1623 विधि है स्पर्शरेखा फ्लो ultrafiltration खोखले फाइबर और हीट हदबंदी कदम का उपयोग जलजनित पता लगाने<em&gt; Cryptosporidium</em&gt; और<em&gt; Giardia एसपीपी.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

अनुभवहीन सीडी 4 की adenoviral पारगमन Treg भेदभाव का अध्ययन करने के लिए कोशिकाओं टी

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

एक प्रतिजन संचालित कोलाइटिस मॉडल के विकास के एंटीजन टी कोशिकाओं को प्रकोष्ठों पेश करके प्रतिजनों की प्रस्तुति का अध्ययन करने के लिए

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

प्रेसिजन कटौती माउस फेफड़े स्लाइस लाइव फेफड़े वृक्ष के समान कोशिकाओं कल्पना करने के लिए

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

रैपिड न्युट्रोफिल केमोटाक्सिस के लिए एक ऑल-ऑन-चिप विधि रक्त के एक बूंद से सीधे विश्लेषण

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

ओवेरेक्स्पेशन एंड हेरिंग ऑफ ह्यूमन<em&gt; सीआईएस</em&gt; -प्रेनिलट्रांसफ़्रेज़ इन<em&gt; एस्चेरिचिया कोली</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

एक उपंयास फीडर इन विट्रो में Murine प्राकृतिक हत्यारा कोशिकाओं के बड़े पैमाने पर उत्पादन के लिए मुफ्त प्रणाली

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

विकास और एक Ultrasensitive एकल अणु सरणी डिजिटल एंजाइम के सत्यापन-मानव इंटरफेरॉन के लिए Immunosorbent परख से जुड़े-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...