ברצוננו להודות לד&quot;ר פול ווייגל על ​​אונ. אוקלהומה לשימושם של נוגדנים חד שבטיים 30 לגילוי של הקולטן HARE/Stabilin-2 וג&#39;נט ווייגל לקבלת סיוע טכני שלה. מחקר זה מומן על ידי אוניברסיטת נברסקה קרנות מחקר.

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Res. 99, 444-449 (1976).</li><li>Graupera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinusoidal+endothelial+COX-1-derived+prostanoids+modulate+the+hepatic+vascular+tone+of+cirrhotic+rat+livers.">Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 288, 763-770 (2005).</li><li>Yannariello-Brown, J., Zhou, B., Ritchie, D., Oka, J. A., Weigel, P. H. <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+ligand+blot+assay+detects+different+hyaluronan-binding+proteins+in+rat+liver+hepatocytes+and+sinusoidal+endothelial+cells.">A novel ligand blot assay detects different hyaluronan-binding proteins in rat liver hepatocytes and sinusoidal endothelial cells.</a> Biochem. Biophys. Res. 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אין ניגודי אינטרסים הכריז.

הרדמה של החולדה בשיטה טיפה תלוי בו isoflurane הכרה אדי מעוררת היא השיטה המועדפת ללימודים שלנו. מאדה עשוי לשמש גם במקום מזרק עם צמר גפן כדי לשמור על חיים מחוץ לחדר, אך ניתוח זה כל כך מהר, זה לא נדרש. פוליאתילן גליקול מתווספת isoflurane כדי להקטין את לחץ אדים וקצב אידוי. יותר מדי אדי isoflurane תגרום למוות מהר מדי. אם החיה מתה לפני הכבד cannulated והחוויר עם TBS, איגום קריש דם עלול בכבד ולמנוע כביסה יעיל של sinusoids ועיכול עם collagenase. תוספת של הפרין עשוי להיות שימושי כמו קרישה, אך אינו משמש במחקרים אלה שכן אנו משתמשים הפרין שכותרתו בדיקה שלנו. שנאגרו של גיי תמיסת מלח (GBSS) מוחלף לעתים קרובות לטיהור של שניות על ידי מעבדות אחרות. אמנם זה שימושי עבור טיהור-SEC, בענייניםatocytes לא לסבול GBSS וכן Buffers 1-3 ו viabilities אינם בגובה דומה. כאשר מודדים אנדוציטוזה, זהו תרגול טוב כדי למדוד את רמות הרקע האיבר וגם מטוהרים hepatocytes. שיטה זו היא אחת משתי לטיהור יעיל של שניות לאחר זלוף כבד עם collagenase. השיטה האחרת מפרידה הלא parenchymal תאים על ידי צנטריפוגה elutriation 19 במקום שיפוע Percoll. היתרונות לשימוש elutriation על הדרגתיים Percoll הם viabilities תא מעט גבוה יותר ומספרים. החיסרון הוא צנטריפוגה elutriation דורש הרוטור התמחה, צנטריפוגה ייעודי, ומשאבה peristaltic יחד שעולה עשרות אלפי דולרים. שיטה זו רק מחייב שימוש צנטריפוגה העליון בקירור שולחן המשאבה peristaltic אשר תקן במעבדות רבות. הפרדת שניות ו KCs על ידי הדבקה סלקטיבית היא שיטה סטנדרטית20-22. אמנם נכון כי שניות תדבק זכוכית ופלסטיק, נקודת המפתח היא להשתמש חומצה שטף זכוכית נקייה עם לא יותר מ דגירה של 15 דקות בשעה 37 ° C 5% CO 2. KCs תמיד לדבוק שניות מהר יותר וגם רצוי לשטוף בעדינות את KCs עם התקשורת כדי לחלץ כל שנותר שניות שהפכו מחוברת באופן רופף. Pronase ו פרוטאזות אחרים השמיט גם את מערכת העיכול צעדים collagenase בפרוטוקול זה כדי להגדיל את viabilities של התאים. פרוטאזות לעיכול קולטני תאי ועלול לעכב הידבקות הסלולר השלבים לטיהור הסופי. השיטה המוצגת כאן יש מגוון רחב של יישומים. למרות שאנחנו מראים את התוצאה הסופית שבה הפרין נלקח בתחילה ל זלוף אישור, הכבד להשגת hepatocytes העיקרי, KC, שניות, ועל HSCs עשוי להיות שימושי עבור מספר מחקרים שכללו מסלולים מטבוליים, immunoregulation, פעילויות נבלות, ועוד פיזיולוגיים Studies. החלק הקשה ביותר של הליך זה הוא cannulation טוב, עיכול של הכבד, וטיפול בכבד מבלי להחליק צנתר מהעורק הפורטל.

<ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מאגר 1: 142 mM NaCl 6.7 mM KCl, HEPES 10 מ&quot;מ, 1.5% BSA, pH 7.4 </li><li style=";text-align:right;direction:rtl"> מאגר 2 (חיץ זלוף): 67.0 mM NaCl, KCl mM 6.7, 4.8 mM CaCl 2-H2O, HEPES 101.0 מ&quot;מ, BSA 1.5%, pH 7.4. </li><li style=";text-align:right;direction:rtl"> מאגר 3: 137 mM NaCl, KCl mM 4.7, 0.7 mM MgSO 4, 1.2 mM CaCl 2-2H 2 O, HEPES 10 מ&quot;מ, 1.5% BSA, pH 7.4 </li><li style=";text-align:right;direction:rtl"> RPMI / BSA: 15g / L BSA ב RPMI </li><li style=";text-align:right;direction:rtl"> טריס שנאגרו מלוחים (TBS): 154 mM NaCl, 10 mM מלח Trizma Base. </li><li style=";text-align:right;direction:rtl"> Collagenase: collagenase (# 11088785103) של חברת רוש, collagenase Type I (# LS004691) מ וורטינגטון ביוכימיים או collagenase אני (# C0130) או IA (# C9891) מבית Sigma-Aldrich. </li></ul> כל המלחים מן Sigma-Aldrich <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> דגם / קטלוג # </td><td> הערה </td></tr><tr><td> אמבט מים 20L </td><td> ThermoFisher </td><td> 2231 </td><td> המים על 45 מעלות צלזיוס, כדי לפצות על קירור </td></tr><tr><td></td><td></td><td></td><td> בתוך הצינור. </td></tr><tr><td> Peristaltic משאבה </td><td> קולמן בן הזוג </td><td> 7553-70 </td><td> Masterflex סדרה </td></tr><tr><td> קירור צנטריפוגה </td><td> Sorvall </td><td> מקרא XTR </td><td></td></tr><tr><td> צנתר </td><td> BD Biosciences </td><td> 381444 </td><td></td></tr><tr><td> Dessicator קאמרית </td><td> דיג </td><td> 08595E </td><td> השתמש צלחת פנימי </td></tr><tr><td> Percoll </td><td> סיגמא </td><td> P4937 </td><td></td></tr><tr><td> שור סרום אלבומין </td><td> SeraCare </td><td> AP-4510-01 </td><td></td></tr><tr><td> 100 מיקרומטר רשת </td><td> ספקטרום מעבדות </td><td> 146488 </td><td></td></tr><tr><td> 30 מיקרומטר רשת </td><td> ספקטרום מעבדות </td><td> 146506 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870 </td><td></td></tr><tr><td> פוליאתילן גליקול </td><td> ספקטרום מעבדהs </td><td> PO107 </td><td></td></tr></tbody></table>

in vivo liver endocytosis followed by purification of liver cells by liver perfusion

הכבד הוא מרכז חילוף החומרים בגופם של יונקים ומשמש כמסנן עבור בדם. הארכיטקטורה הבסיסית של הכבד מתוארת באיור 1, שבו יותר מ 85% ממסת הכבד מורכב hepatocytes ואת 15% הנותרים של מסת הסלולר מורכב של תאים Kupffer (KCs), בתאי stellate (HSCs), ו לתאי אנדותל סינוסי (שניות). שניות בצורת דפנות כלי הדם בתוך הכבד מכילים מורפולוגיה מיוחד הנקרא fenestrae בתוך בציטופלסמה. אשנוב של הציטופלסמה היא מראה של חורים (~ 100 מיקרומטר) בתוך התאים, כך לפעול שניות כמו מסננת שבה רוב chylomicrons, שרידי chylomicron ו מקרומולקולות, אך לא תאים, עוברים את hepatocytes ו HSCs 1 (איור 1). בשל היעדר קרום במרתף, הפער בין שניות ו hepatocytes בצורת שטח של Disse. HSCs למלא חלל זה ולשחק תפקיד מרכזי בוויסות ותגובה injury, אחסון של החומצה הרטינואית ו immunoregulation 2 של הכבד. שניות הם בין התאים הפעילים ביותר בגוף endocytically הצגת מערך של קולטנים נבלות על פני התא שלהם 3. אלה כוללים SR-A, Stabilin-1 ו Stabilin-2. באופן כללי, חלקיקים colloidal קטן פחות מ 230 ננומטר מקרומולקולות בשלב חיץ נתפסות על ידי שניות, ואילו, חלקיקים גדולים ופסולת הסלולר הוא endocytosed (phagocytosed) על ידי KCs 4. לפיכך, אישור עיקר החומר תאיים כגון glycosaminoglycans מהדם תלויה במידה רבה על הבריאות פונקציות endocytic סעיפים 5,6. לדוגמה, עלייה בדם hyaluronan מעיד על מחלת כבד החל קלה עד צורות חמורות יותר 7. למעט דו&quot;ח אחד 8, אין קווי הנציח SEC תא הקיום. אפילו את שורות התאים הוא הנציח דה differentiated בכך שהוא אינו מבטא קולטנים נבלות שנמצאים שניות הראשי (הנתונים שלנו, לא מוצג). מחקרים ביולוגיים כל תא חייב להתבצע על תאים ראשוניים שהתקבלו טרי מן החיה. לרוע המזל, שניות dedifferentiate בתנאים תרבות סטנדרטי יש להשתמש תוך 1 או 2 ימים על במנותק מן החיה. דיפרנציאציה של שניות מסומן על ידי הביטוי של Stabilin-2 או קולטן, הארי 9, CD31, ואת הנוכחות של אשנוב cytoplasmic 1. דיפרנציאציה של שניות ניתן להאריך על ידי תוספת של VEGF בתקשורת תרבות או על ידי תאים culturing במדיום מותנה hepatocyte 10,11. בדו&quot;ח זה, נדגים את הפעילות endocytic של שניות בתוך איבר שלם באמצעות רדיו שכותרתו הפרין עבור hyaluronan עבור קולטן Stabilin-2-SEC ספציפיים. לאחר מכן אנו לטהר hepatocytes ו שניות מהכבד perfused למדוד אנדוציטוזה.

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In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

המחקר בתאי הכבד סינוסי אנדותל (שניות) חייב להתבצע עם תאים ראשוניים שהתקבלו החיה לא כמו שורות תאים קיימות. שיטה זו מסתמכת על מערכת העיכול הכבד צנטריפוגה ההפרש לטיהור SEC עבור culturing ניסויים עתידיים.

ב אנדוציטוזה כבד vivo ואחריו טיהור בתאי הכבד על ידי זלוף כבד

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

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In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

18.9K Views.  University of Nebraska, Lincoln. המחקר בתאי הכבד סינוסי אנדותל (שניות) חייב להתבצע עם תאים ראשוניים שהתקבלו החיה לא כמו שורות תאים קיימות. שיטה זו מסתמכת על מערכת העיכול הכבד צנטריפוגה ההפרש לטיהור SEC עבור culturing ניסויים עתידיים.

Video: In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

בידוד של הכבד CD133 + בתאי גזע משובטים עבור הרחבת

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

הבידוד של Fibroblasts פורטל עכברוש ידי<em&gt; באתרו</em&gt; כבד זלוף

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

עשה זאת בעצמך התקן להבראת דוגמאות Cryopreserved בטעות ירד לתוך טנקים אחסון קריוגני

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

בידוד הכרומטין על ידי טיהור RNA (ציוץ)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

בידוד של hepatocytes האנושי על ידי שני שלבים Collagenase זלוף נוהל

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

במעקב 4 ממדי Vivo של תאי גזע ואבות היווצרות דם במבוגרי עכבר calvarial מח עצם

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

פרוטוקול לlentiviral התמרה וניתוח במורד הזרם של המעיים Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

סטנדרטי שיטה לניתוח של תאי הכבד סינוסי האנדותל וFenestrations על ידי סריקה מיקרוסקופית אלקטרונים

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

ניתוח Glucocorticoid צואה: לא פולשני ניטור הכליה ב Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

סוג תא-ביטוי גנים ספציפי ליצירת פרופיל בכבד העכבר

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...