Collagenase הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תשקלי את collagenase לתוך חיץ 50 מ&quot;ל בידוד חרוטי ולהוסיף להפוך את הריכוז הסופי, כמפורט בטבלה שלהלן. ממיסים את collagenase ידי vortexing. (12 + 2 עכברים עכברים עכברים תוספת = 14 x 5 מ&quot;ל / עכבר = 70 מ&quot;ל collagenase פתרון מוחלט) (70 מ&quot;ל x 0.5 מ&quot;ג / מ&quot;ל ​​או 0.3 מ&quot;ג / מ&quot;ל ​​= 35 מ&quot;ג או 21 מ&quot;ג בסך P collagenase) <table style="BORDER-RIGHT: #ddd 1px solid; BORDER-TOP: #ddd 1px solid; BORDER-LEFT: #ddd 1px solid; BORDER-BOTTOM: #ddd 1px solid; TEXT-ALIGN: left;text-align:right;direction:rtl" border="0" width="80%"><tbody><tr><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left"> זן </th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left"> גיל </th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left"> Collagenase P (מ&quot;ג / מ&quot;ל) </th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left"> זמן (דקות) </th></tr><tr><td style="font-size: 12px"> C3H, Balb / C, B6 </td><td style="font-size: 12px"> &gt; 12 שבועות </td><td style="font-size: 12px"> 0.8 </td><td style="font-size: 12px"> 17 </td></tr><tr><td style="font-size: 12px"> C3H, Balb / C, B6 </td><td style="font-size: 12px"> &lt;= 12 שבועות </td><td style="font-size: 12px"> 0.5 </td><td style="font-size: 12px"> 13 </td></tr><tr><td style="font-size: 12px"> מהנהנת, NOR, אי </td><td style="font-size: 12px"> &gt; 12 שבועות </td><td style="font-size: 12px"> 0.8 </td><td style="font-size: 12px"> 17 </td></tr><tr><td style="font-size: 12px"> מהנהנת, NOR, אי </td><td style="font-size: 12px"> &lt;= 12 שבועות </td><td style="font-size: 12px"> 0.5 </td><td style="font-size: 12px"> 17 </td></tr></tbody></table> </li><li style=";text-align:right;direction:rtl"> לוותר על 2 מ&quot;ל של תמיסת collagenase לתוך בקבוקון זכוכית בכל מקום על הקרח. טען כל מזרק 5 מ&quot;ל עם 3 מ&quot;ל של מחט collagenase להוסיף 30G מקום בקרח (להכין במנדף). </li></ol> Distension <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> רגע לפני לנתיחה, להקריב בעלי חיים על ידי נקע בצוואר הרחם. ספריי על כל הגוף עם 70% ETOH, מה שהופך אותו רטוב לגמרי. הפוך V-insision החל באזור אברי המין. סובב את העכבר כך הזנב פונה ממך. </li><li style=";text-align:right;direction:rtl"> הסר את המעי לצדו השמאלי של העכבר פתוח. זה יחשוף את הלבלב צינור המרה המשותף. </li><li style=";text-align:right;direction:rtl"> מניחים מהדק hemostat משני צדי קטן המעי, שם את צינור המרה מנקז, משאיר כיס קטן לפתרון collagenase להזין את המעי. </li><li style=";text-align:right;direction:rtl"> לנפח את הלבלב דרך בצינור המרה עם מחט 30G ו 5 מ&quot;ל במזרק המכיל 3 מ&quot;ל של תמיסת collagenase קר, החל בכיס המרה. </li><li style=";text-align:right;direction:rtl"> הסר את הלבלב מהגוף ולמקם אותו בקבוקון siliconized המכיל 2 מ&quot;ל של תמיסת collagenase. צעד זה צריך להיעשות במהירות ככל נקי ככל האפשר, מזעור אוסף של רקמות שומן החיבור. </li><li style=";text-align:right;direction:rtl"> מניחים את הצנצנת על קרח שלב 3 לחזור עם העכבר הבא. </li></ol> עיכול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותם כל בקבוקון ולמקם אותו לתוך אמבט 37 ° C-המים. דגירה של 13-17 דקות (הזמן משתנה עם הגיל זן של בעל החיים). </li><li style=";text-align:right;direction:rtl"> לאחר זמן שחלף, לנער את הבקבוקון במרץ. הלבלב צריך להתפרק. </li><li style=";text-align:right;direction:rtl"> יוצקים כל לעכל דרך מסננת כיור גדול סטרילי לתוך מבחנה סטרילית 1000 מ&quot;ל ו - בכוח פיפטה את המסך עם כביסה חיץ. לוותר מעכל לתוך צינורות conicals 50 מ&quot;ל (3 pancreata/50 צינור מ&quot;ל: אם n = 12, השתמש מספיק כביסה חיץ כדי להפוך את סופי 200 מ&quot;ל נפח ומוציאים אותו לצינורות 4x50 מ&quot;ל). דילול צריך להיעשות במהירות על הקרח, כדי למנוע עיכול איון לא רצויות. </li><li style=";text-align:right;direction:rtl"> ספין למטה: הפעל את הצנטריפוגות. כאשר זה מגיע 1300 סל&quot;ד, לכבות אותו. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant, עוזב ~ 5 מ&quot;ל. היה זהיר מאוד שלא לשאוב את הכדור. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה ידי הקשה נמרצות עם היד שלך, ולאחר מכן להוסיף 50 מ&quot;ל של חיץ לשטוף. </li><li style=";text-align:right;direction:rtl"> ספין למטה: הפעל את הצנטריפוגות. כאשר זה מגיע 1300 סל&quot;ד, לכבות אותו. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה ולשטוף עם 5 מ&quot;ל של חיץ כביסה. </li><li style=";text-align:right;direction:rtl"> העברת מ&quot;ל 5-15 מ&quot;ל חרוטי ספין למטה, כאמור לעיל. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant כפי לחלוטין ככל האפשר. (חיץ נותרת עלול לגרום שינוי צפיפות של Ficoll). </li></ol> Ficoll בצע במהירות. חשיפה ארוכת טווח Ficoll רעיל איים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא גלולה שבור לגמרי לפני הוספת ficoll (רקמה רצוף קשה resuspend ב ficoll). </li><li style=";text-align:right;direction:rtl"> Resuspend את הכדור בתוך 7 מ&quot;ל של ficoll, צפיפות 1.108, על ידי vortexing במרץ. </li><li style=";text-align:right;direction:rtl"> שכבה על גבי צפיפות כל ficoll 2ml של כל אחד צפיפויות הנותרים לפי הסדר הבא: 1.096, 1.069, 1.037 אז. </li><li style=";text-align:right;direction:rtl"> ספין במשך 15 דקות ב 1800 סל&quot;ד ב 4 מעלות, עם לנתק! </li><li style=";text-align:right;direction:rtl"> פיק איים מן השכבה השנייה בעזרת spoid (p סטריליתהטפטפת lastic). העברת כל האוספים ל 50 מ&quot;ל המכיל 25 מ&quot;ל חרוטי ~ של חיץ קר. </li><li style=";text-align:right;direction:rtl"> שטפי, כאמור לעיל, 3 פעמים עם חיץ כביסה (חוזרת צעד 12-15). </li><li style=";text-align:right;direction:rtl"> איי Resuspend ב 20 מ&quot;ל RPMI-1640 (FCS המכיל 10% ו פניצילין וסטרפטומיצין, HEPES, מ-NEAA) ומערבבים בעדינות. הסר ul 100 המדגם עבור לספור. </li><li style=";text-align:right;direction:rtl"> העברת 100 ul עד 35 צלחת רשת מ&quot;מ המכילה 1 מ&quot;ל של מדיה 1 dithizone מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> דגירה איים שנותרו RPMI 1640, ב 37 ° C, 7% CO 2 באינקובטור, ב 160 מ&quot;מ צלחות עם סך של 30 מ&quot;ל של מדיה / צלחת. <img src="/files/ftp_upload/old/255_2008_8_24_3/255_Figure-01.png" alt="איור 1" width="220" height="194" /></li></ol>

murine pancreatic islet isolation

Watch this Scientific Journal Video about Murine Pancreatic Islet Isolation at JoVE.com

Murine Pancreatic Islet Isolation

הלבלב Murine איילט בידוד

Research

JoVE Journal

Biology

38.4K Views.  University of California, San Francisco - UCSF. 

Video: Murine Pancreatic Islet Isolation

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

בידוד האדם לבלב: אני חלק: העיכול אוסף של רקמת לבלב

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

הלבלב האנושי בידוד איילט: חלק ב: טיהור והתרבות של איי האדם

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

בידוד של בתאי גזע xenografts סרטן הלבלב האנושי

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

בסיוע מחשב ויזואליזציה בקנה מידה גדול כימות המוניים הלבלב איילט, התפלגות גודל ואדריכלות

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

העכבר איילט בידוד נגרהנס באמצעות שילוב של collagenase המטוהר ופרוטאז הניטראלי

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

שיטה לבידוד עכבר הלבלב איילט וקביעת תאית cAMP

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

בידוד והתרבות של תאי לבלב הראשיים acinar עכבר

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

בידוד של תאי Murine Valve האנדותל

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

בידוד של תאי שריר חלק Murine כלילית דם

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.