TMS נתמך על ידי מלגת הרלן הנאמנות (GWU) במהלך מחקר זה. מימון לאוסף שדה ורביית L. fasciola וא delphinii סופק על ידי מענק NSF DEB 0642438 לJTL.

1. הכנת חומר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את המחטים (1-2 קצה מיקרומטר ולהתחדד מ&quot;מ 3-4) באמצעות צינורות זכוכית נימי ורוסיליקט וחולץ micropipette. הגדרות מכשיר: רמפה: 561; מהירות: 20; חום: 560; עיכוב: 1; משיכה: 100; לחץ: 500. </li><li style=";text-align:right;direction:rtl"> הכן את פתרון האוסף: 60% מבינוניים של גרייס (GM) בתוספת 10% בסרום שור עוברי (FBS) ו 20% ממאגר נוגד קרישה (98 mM NaOH, 186 המ&quot;מ NaCI, 1.7 mM EDTA וחומצת לימון 41 מ&quot;מ, pH 4.5 ). הפתרון לעיל מוכן טרי בתנאים סטריליים ושמר על קרח בכל העת. </li><li style=";text-align:right;direction:rtl"> הכן את פתרון חיץ דגירה: 90% מGM בתוספת 10% FBS. פתרון זה הוא גם מוכן טרי ושמר על קרח בכל העת. </li><li style=";text-align:right;direction:rtl"> הכן כולל של לפחות 15 μl × מספר החרקים עבור שניהם אוסף ואת פתרוני הדגירה. </li></ol> 2. הזרקה של זחלים עם פתרון אוסף <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרדם תוספות הלפני אחרונותזחלי זפת ידי מצמרר אותם בקרח למשך 20 דקות לפני חילוץ hemocyte. </li><li style=";text-align:right;direction:rtl"> התאם את הצד הקהה ​​של מחט נימית נקיה (16-מד) לתוך הצינור שמחובר למזרק זכוכית מ&quot;ל 20 (איור 2). השאר את המבחנה המכילה תמיסת האוסף לפתוח על ספסל המעבדה ולמלא את הנימים לאט עם החיץ. הנימים בדרך כלל מלאות בכ 10-15 μl של פתרון אוסף, בהתאם לגודלו של הזחל (כ 1 פתרון μl ל10 מ&quot;ג של מסת גוף כוללת זחל = 0.01% ממסת גוף הכוללת). הנח את המחט על כדור הטיט הממוקם בסמוך למיקרוסקופ לנתח. </li><li style=";text-align:right;direction:rtl"> הנח את הזחל בצלחת פטרי. החרק צריך להיות ממוקם בצד שלה, כך שהם נראים בבירור spiracles (איור 3). </li><li style=";text-align:right;direction:rtl"> במהלך ההצגה תחת מיקרוסקופ לנתח, החזק הזחל במקום עם זוג מלקחיים ולמקם את המחט הקרובה לאחד מspiracles, שם בציפורן כת היא בדרך כלל רכה יותר. לחץ בעדינות על מחט הנימים נגד הגוף של החרק. ברגע שהמחט חדרה לציפורן, להזריק נפח הכולל של תמיסה לתוך הזחל (איור 3). חשוב להימנע מלהזריק בועות אוויר לתוך הזחל. גופו של הזחל שיתנפח (למשל, גידול בהיקף של 20%) לאחר הזרקה אבל לא צריך להתפוצץ או לטפטף. </li><li style=";text-align:right;direction:rtl"> להחזיר את הזחל לקרח, מיצוב חרקי dorsally חיץ כדי למנוע דליפה מהפצע (איור 3). </li><li style=";text-align:right;direction:rtl"> חזור על שלבים 2.1-2.4 לכל זחל משמש להפקת hemolymph. בשלב זה, לאפשר לכל החרקים לצנן על קרח למשך 30 דקות לפני שתמשיך. </li></ol> 3. הפקת Hemocytes <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח את הזחל בצלחת פטרי תחת מיקרוסקופ לנתח. </li><li style=";text-align:right;direction:rtl"> עושה חתך רדוד (2-3 מ&quot;מ אורך) באזור האחורי של הזחל באמצעות scalpe סטרילי<html> l (איור 3). הקפד שלא לפגוע במעיים או האיברים פנימיים אחרים; המטרה היא לנקב את הציפורן בזהירות ולאסוף את תמהיל החיץ / hemolymph מכיל hemocytes מחרקים בודדים. </li><li style=";text-align:right;direction:rtl"> לסחוט את הזחל בעדינות באמצעות מלקחי פלסטיק גמישים תוך איסוף תמהיל hemolymph (כ 10-20 μl) עם קצה פיפטה 10 μl, שוב נזהר שלא לפגוע ברקמות הפנימיות (איור 3). Hemolymph נאסף מהאגל לחץ מגופו של הזחל. השלך שרידים של הזחל. </li><li style=";text-align:right;direction:rtl"> איסוף hemolymph מכל זחל פרט ל0.5 צינורות נפרדים שכותרת siliconized מיליליטר microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף נפח שווה של פתרון הדגירה (כ 10-20 μl) לכל דגימת hemolymph נאספת מזחל פרט. שמור דגימות hemolymph על קרח בכל העת. </li><li style=";text-align:right;direction:rtl"> חזור על שלבים 3.1-3.5 לכל זחל השתמש בניסוי. </li></ol>&quot;&gt; 4. כימות le של Hemocytes <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מערבב את מדגם hemolymph ופתרון הדגירה מייד לפני hemocytes ספירה. טיפול זה מאפשר הפרדה נכונה של hemocytes ומונע יצירת גושים. </li><li style=";text-align:right;direction:rtl"> השתמש בקצה פיפטה נקי להעביר 10 μl של מדגם hemolymph מעורבב עם פתרון הדגירה על hemocytometer. </li><li style=";text-align:right;direction:rtl"> באמצעות מיקרוסקופ מתחם, לספור ולרשום את המספר של hemocytes בלפחות 15 הריבועים הגדולים (שטח של ריבוע אחד: 0.04 מ&quot;מ 2, עומק: 0.1 מ&quot;מ) של הרשת המרכזית של hemocytometer (איור 3). הוסף ערכים אלה לקבל סכום כולל של מספר hemocytes ב15 הריבועים. </li><li style=";text-align:right;direction:rtl"> שטוף את hemocytometer ושקופית העטיפה שלו ביסודיות באמצעות בקבוק לשטוף במים מזוקקים וdeionized ולנגב יבש עם Kimwipes (קימברלי קלארק) בין כל דגימה. </li><li style=";text-align:right;direction:rtl"> חזור על שלבים 4.1-4.4 לכל דגימת hemolymph. </li><li style=";text-align:right;direction:rtl"> חישוב המספר הממוצע של hemocytes למ&quot;ל של hemolymph volumדואר לכל זחל, כדלקמן: </li></ol> נפח כולל של כל ריבוע של רשת hemocytometer: (0.04 2 אזור מ&quot;מ) × (0.1 מ&quot;מ עומק) = 0.004 3 מ&quot;מ × 15 ריבועים = 0.06 מ&quot;מ 3 = נפח הכולל של כל 15 הריבועים נספרו. (סכום של המספר הכולל של hemocytes ב15 הריבועים נספרו / הנפח הכולל של כל 15 הריבועים נספרו) x 2 = גורם דילול מספר hemocytes / המ&quot;מ x 3 1000 = מספר hemocytes / המ&quot;ל של hemolymph.

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שיטות לחילוץ hemocytes מחרקים רפואיים חשובים וחרקי מודל lepidopteran העבר דיווח 9, 14. שיטות חילוץ Hemocyte מותאמות על פי מיני חרקים, השלב ההתפתחותי של החרק ותכונות המורפולוגיות שלה. לדוגמה, בידוד hemolymph מזחלי Manduca יכול להתבצע בקלות על ידי חיתוך הקרן המעוקלת סמוך לסוף הבטן <s...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> Micropipette חולץ </td><td> סאטר מכשירים </td><td> P-1000 </td><td></td></tr><tr><td> צינורות זכוכית ורוסיליקט </td><td> סאטר מכשירים </td><td> B100-50-10 </td><td> OD: 1.0, מזהה: 0.50 מ&quot;מ </td></tr><tr><td> הבינוני של גרייס </td><td> סיגמא </td><td> G8142 </td><td></td></tr><tr><td> סרום שור עוברי </td><td> תרם הפישר סיינטיפיק </td><td> SH3007102 </td><td> חום מומת </td></tr><tr><td> דומון # 5 מלקחיים </td><td> כלי המדע פיין </td><td> 11252-40 </td><td></td></tr><tr><td> נויבאואר hemocytometer </td><td> אוסר מדעי </td><td> 3200 </td><td></td></tr><tr><td> פלסטיק טוביםng </td><td> Tri-Tech </td><td> TT-3-32OD </td><td> OD: 3/32&#39;&#39;, מזהה: 1/32 &#39; </td></tr><tr><td> זכוכית רפואית מזרק </td><td> פורטונה אופטימה </td><td> D-97877 </td><td> 50 מיליליטר נפח </td></tr><tr><td> מחט קצה קהה </td><td> חלקים קטנים </td><td> NE-162PL-25 </td><td> 16 מד x 1 &quot;אורך </td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

hemocytes חרקים (שווה ערך לתאי דם לבנים יונקים) משחק תפקיד חשוב בכמה תהליכים פיסיולוגיים לאורך כל מחזור החיים של חרק 1. בשלבי זחל של חרקים השייכים לצווים של פרפרים (עשים ופרפרים) וDiptera (זבובים אמיתיים), hemocytes נוצרים מבלוטת הלימפה (hematopoietic איבר מיוחד) או התאים עובריים ויכולה להתבצע דרך לשלב הבגרות. hemocytes העוברי מעורב בנדידת תאים במהלך התפתחות הרגולציה וchemotaxis במהלך דלקת. הם גם ייקחו חלק באפופטוזיס תא והם חיוניים להתפתחות עוברת 2. Hemocytes לתווך זרוע התאית של תגובת החיסון המולדת חרקים שכוללת מספר פונקציות, כגון תא, צבירה מתפשטת תא, היווצרות של גושים, phagocytosis ואנקפסולציה של פולשים זרים 3. הם גם אחראים לניהול הגנה הומורלית חרקים מסוימות במהלך זיהום, כגון יחסי הציבורoduction מיקרוביאלית של פפטידים ומולקולות אחרות מפעיל 4, 5. המורפולוגיה והתפקוד Hemocyte שנחקרו בעיקר במודלים של חרקים גנטיים או פיסיולוגיים, כוללים זבוב פרות, דרוזופילה melanogaster 6, 7, יתושי Aedes aegypti ואנופלס gambiae 8, 9 והטבק hornworm, Manduca 10 סקסטה, 11. עם זאת, מעט מידע קיים היום על הגיוון, סיווג, מורפולוגיה והתפקוד של hemocytes במינים שאינם מודל חרקים, במיוחד אלה שנאספו מ12 הפרועים. כאן אנו מתארים פרוטוקול פשוט ויעיל לחילוץ hemocytes מזחלים פרא. אנו משתמשים instar Lithacodes fasciola הלפני האחרון (עש קליע צהוב כתפיים) (איור 1) וEuclea delphinii (קליע אלון קוצני) זחלים (פרפרים: Limacodidae) ומראים כי היקפים מספיקים של hemolymph (דם חרקים)יכול להיות מבודד ומספרי hemocyte נספרו מזחלי פרט. שיטה זו יכולה להיות מנוצלת ביעילות ללמוד סוגי hemocyte במינים אלה, כמו גם בזחלי lepidopteran נלווים אחרים שנקטפו מהשדה, או שזה יכול להיות בקלות בשילוב עם מבחנים אימונולוגיים שנועדו לחקור את תפקוד hemocyte בעקבות הידבקות בחיידקים או אורגניזמים טפילים 13.

הפרוטוקול המתואר כאן מאפשר גביית היקף מינימאלי של 10-20 μl של hemolymph חרקים מזחלים בודדים. Hemocytes שנאסף בשיטה זו היא ללא clumping תא, פגמי melanization, פסולת רקמה או מזהמים אחרים. לכן hemocytes ניתן להבחין בקלות וספר מתחת למיקרוסקופ, וכמה חרקים ניתן להבחין בתוך כמה Hosur. הבחנו כי רוב hemocytes ב?...

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A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

hemocytes חרקים למלא תפקידים חשובים רבים, הוא חיסון ושאינם מחוסנים, לאורך כל שלבי התפתחות חרקים. הידע הנוכחי שלנו של הסוגים והתפקוד hemocyte מגיע ממחקרים על מודלים גנטיים של חרקים. כאן, אנו מציגים שיטה לחילוץ, כימות והמחשת hemocytes מזחלים פרא.

פרוטוקול פשוט לחילוץ Hemocytes מזחלי Wild

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

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Research

JoVE Journal

Biology

16.1K Views.  The George Washington University. hemocytes חרקים למלא תפקידים חשובים רבים, הוא חיסון ושאינם מחוסנים, לאורך כל שלבי התפתחות חרקים. הידע הנוכחי שלנו של הסוגים והתפקוד hemocyte מגיע ממחקרים על מודלים גנטיים של חרקים. כאן, אנו מציגים שיטה לחילוץ, כימות והמחשת hemocytes מזחלים פרא.

Video: A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

פרוטוקול לגידול יתושים (א gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: שיטה פשוטה genotyping מדויק

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

פרוטוקול נוירונים culturing הסימפתטית מן הגרעינים עכברוש צוואר הרחם מעולה (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

פרוטוקול לייצור KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

PCR מבוסס genotyping שיטה להבחין בין זנים wild-type ו נוי של Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

טכניקה פשוטה ויעילה להכנת השעיות תא אשכים

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

SIVQ-LCM פרוטוקול עבור מכשיר ArcturusXT

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

מבדק פשוט עבור הערכת גורמי הגדילה של אנדותל כלי דם

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

שיטה לחילוץ פיגמנטים מן קלמארי<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

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.