המחברים מודים דן ובס מ סיגמא אולדריץ להצגת וידאו. אנו מודים גם דון סניף, Evgenii צ&#39;רנישב, סקוט Chesla, אליזבת Hadac, אמנדה הריסון, דמיאן היתקוט, אניקה Hult, צ&#39;ואן צ&#39;ינג-Lan, סוזנה מקינטוש, Sarvani Komarraju, אלנה Korchagina, קרוליין אוליבר, מרטין אולסון, סטיבן פרקר, איגור Rodinov, אלכסנדר Tuzikov, ואלינור וויליאמס לתוצאות שלהם תרומות סינתזה העיצוב, של מבנים FSL וקביעת הפעילות הביולוגית.

<ol>
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Hematol. 16, 467-472 (2009).</li><li>Oliver, C., Blake, D., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+neutralization+of+anti-A+and+successful+transfusion+of+A+antigen+incompatible+red+cells+in+an+animal+model.">In vivo neutralization of anti-A and successful transfusion of A antigen incompatible red cells in an animal model.</a> Transfusion. , (2011).</li><li>Oliver, C., Blake, D., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+transfusion+reactions+and+predicting+in+vivo+cell+survival+with+kodecytes.">Modeling transfusion reactions and predicting in vivo cell survival with kodecytes.</a> Transfusion. , (2011).</li><li>Heathcote, D., Carroll, T., Wang, J. 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L., Brad Jones, R., Ramkumar, S., Sakac, D., Binnington, B., Henry, S., Lingwood, C. A., Branch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+globotriaosylceramide+analogue+inhibits+HIV-1+infection+in+vitro+by+two+mechanisms.">A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms.</a> Glycobiology. 27, 515-524 (2010).</li><li>Barr, K., Diegel, O., Parker, S., Bovin, N., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function-Spacer-Lipid+(FSL)+constructs+enable+inkjet+printing+of+blood+group+antigens.">Function-Spacer-Lipid (FSL) constructs enable inkjet printing of blood group antigens.</a> FEBS J. 277, 235-235 (2010).</li><li>Lan, C. -. 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דבורה א &#39;בלנק סטיבן מ&#39; הנרי העובדים ובעלי המניות של KODE ביוטק מוגבלת בעל פטנט של הטכנולוגיה KODE. דן ובס הוא עובד של סיגמא אולדריץ, אשר מימנה את הייצור של מאמר זה וידאו.

תאים שינוי ו virions עם בונה FSL היא טכניקה פשוטה מאוד חזקים 3-11. כדי לתאר ולהבחין FSL לבנות תאים שונה ו virions מתאי ללא שינוי, הם מכונים kodecytes ו kodevirions בהתאמה 6-10, אך רק כאשר הקבוצה הציגה תפקודית ניתן להראות להיות נוכח על הממברנה שלהם. כאשר kodecytes מיוצרים עם ריכוזים שונים של מבנים FSL, הם יכולים להיות מכונה על ידי ריכוז של הפתרון FSL לבנות השתמשו כדי ליצור אותם, למשל 15 מיקרוגרם / מ&quot;ל ​​kodecytes, או אם אחד או יותר FSL לאחר מכן נעשה שימוש במונח משולב, לדוגמא: A + ביוטין kodecytes 7,8. כאשר תאים שונים כדי להשוות, לרבות סוג תא בתיאור מומלץ, למשל אדום ביוטין תא kodecytes או kodecytes ביוטין רירית הרחם 7,8. למרות תהליך הכניסה היא מאוד חזקה הכניסה (אמנם עם תעריפים שונים) תתרחש על פני טווח טמפרטורה גדול 4-37 ° C ו בתוך דקות עד שעות, כדי להשיג שליטה הכניסה לשחזור קפדנית של זמן מגע, טמפרטורה, ריכוז FSL ( כולל ניסוח diluent), וכן ריכוז במידה פחותה תא נדרשים. עד היום, כל המבנים FSL ניתן להשתמש כדי לשנות את התאים על ידי טכניקה זהה 4-11, אולם צפוי כי בתנאים מסוימים יהיו נוחים יותר זרימת עבודה ו / או בדרישות הטיפול האופטימלי של התאים / virions להיות שונה . הריכוז המדויק FSL כדי להשיג את האפקט הרצוי צריך להיקבע על ידי המשתמש 4. FSL כתוצאה לבנות תאים שונה / virions בדרך כלל ניתן להשתמש באותו אופן כמו תא virion ללא שינוי / במערכות ביולוגיות או אנליטי 4-11. כמו בונה FSL יהיה לאט elute ממשטחי שונה כאשר במגע עם בתמיסות 7,8, או להיות נצרך על ידי תאים פעילים 11, נושאים אלה יש לקחת בחשבון ביחס לרמת האות או פעילות שהושגו על פני תקופות זמן. Kodecytes יכול להיות קבוע (למשל glutaraldehyde / פורמלין) לאחר הכניסה בתנאי מקבע אינו מכיל שומנים משחררי ממיסים את הקבוצה פונקציונלי תואם מקבע. תאים קבוע לחילופין יכול להיות שונה עם בונה FSL. בנוסף לשינוי של משטחי תאים virion בונה FSL יכול גם להיות חדורים ישירות למחזור הדם של חיות מעבדה גרימת שינוי vivo של תאים במחזור 7 ו לעכב 12 וירוסים, רעלים, 12 ונוגדנים 7. בונה FLS יש גם שימש לקשט ליפוזומים והוא יכול להיות מודפס על גבי משטחי נייר 13, שם הם לשתק, ולאחר מכן יכול לשמש מבחני אבחון. היכולת לשנות בקלות תאים חיים עם מגוון הולך וגדל של בונה FSL צריך להוכיח להיות מחקר שימושי כלי פיתוח לחקר הביולוגיה פני התא.

<img src="/files/ftp_upload/3289/3289table1.jpg" alt="טבלה 1" /> טבלה 1. זמין מסחרית בונה FSL המתואר במאמר זה.

fsl constructs: a simple method for modifying cell/virion surfaces with a range of biological markers without affecting their viability

היכולת לשנות / לדמיין משטחים ביולוגיים, ולאחר מכן ללמוד את התא שונה / virion במגוון במבחנה בסביבות vivo חיונית להשגת תובנה נוספת לתוך הפונקציה של מולקולות ספציפיות או את הישות כולה. מחקרים של שינוי פני השטח ביולוגיים מוגבלים בדרך כלל הנדסה גנטית של האורגניזם או את הקובץ המצורף קוולנטיים של moieties כימית 1,2 שטח פני התא. אולם אלה טכניקות מסורתיות לחשוף את התא המגיבים כימיים, או שהם דורשים מניפולציה משמעותי כדי להשיג את התוצאה הרצויה, מה שהופך אותם מסורבל, והם עשויים גם להשפיע על הכדאיות בשוגג / הפונקציונליות של התא שונה. שיטה פשוטה מזיק לשנות את פני השטח של התאים נדרשת. לאחרונה טכנולוגיה חדשה, טכנולוגיה KODE הציגה מגוון של מבנים הרומן המורכב משלושה מרכיבים: קבוצה ראש פונקציונלי (F), spacer (S) וזנב השומנים (L) ידועים כמו פונקציה ליפידים-Spacer או FSL constructs3. Spacer (S) נבחרה לספק לבנות כלומר מסיסות במים, עדיין יהיה ספונטני ביציבות לשלב קרום. FSL לבנות moieties תפקודית (F) עד כה כוללים מגוון של saccharides כולל קבוצה הקשורות דם הגורמים, חומצות sialic, סוכרים hyaluronan, fluorophores, ביוטין, radiolabels, וכן מגוון של פפטידים 3-12. בונה FSL שימשו עוברים, זרע שינוי, דג הזברה, לתאי האפיתל / רירית הרחם, כדוריות דם אדומות, ו virions ליצור בקרות איכות מערכות ולוחות אבחון, לשנות הדבקה תא / אינטראקציה / הפרדה / immobilization, ועל במבחנה vivo הדמיה של תאים / virions 3-12. התהליך של שינוי תאים / virions הוא גנרי ופשוט ביותר. ההליך הנפוץ ביותר הוא הדגירה של תאים (בתקשורת חינם שומנים בדם) עם פתרון עבור FSL בונה במשך 1-2 שעות ב 37 ° C 40-10. במהלך הדגירה FSL המבנים באופן ספונטני לשלב הממברנה, והתהליך הושלם. כביסה היא אופציונלית. תאים שונה על ידי בונה FSL ידועים כמו kodecytes 6-9, בעוד virions הם kodevirions 10. FSL בונה כמו חליטות ישיר kodecytes / kodevirions נעשה שימוש במודלים של בעלי חיים ניסיוניים 7,8,10. כל kodecytes / kodevirions להופיע כדי לשמור על חיוניות הרגיל שלהם ואת הפונקציונליות תוך השגת את הפונקציה החדשה של מחצית F 7,8,10,11. השילוב של dispersibility בתקשורת ביולוגית, התאגדות ספונטנית לתוך קרום התא, וכן רעילות נמוכה נראית לעין, שעושה FSL בונה כלי מחקר חשוב למחקר של תאים virions.

Watch this Scientific Journal Video about FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability at JoVE.com

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

פונקציה-Spacer-ליפידים (FSL) בונה לאפשר את תכונות פני השטח של תאים חיים virions להיות שונה ללא אובדן חיוניות. השיטה דורשת מגע פשוט רק פתרון לבנות FSL עם תא / virion ושילוב משטח יציב ספונטנית מתרחשת.

FSL בונה: שיטה פשוטה שינוי Cell / Virion משטחים עם מגוון רחב של סמנים ביולוגיים מבלי להשפיע ליכולת הקיום שלהם

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential ...

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Research

JoVE Journal

Biology

13.7K Views.  AUT University and KODE Biotech Ltd. פונקציה-Spacer-ליפידים (FSL) בונה לאפשר את תכונות פני השטח של תאים חיים virions להיות שונה ללא אובדן חיוניות. השיטה דורשת מגע פשוט רק פתרון לבנות FSL עם תא / virion ושילוב משטח יציב ספונטנית מתרחשת.

Video: FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

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 ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

יישום של C. elegans Assay ניוון Neuron דופמין עבור אימות של מחלת פרקינסון גנים אפשריים

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

המצאה של Myogenic בונה רקמות מהונדסות

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

שיטה לקטינים HPLC להעשיר פפטידים סלקטיבי-glycosylated מ דגימות ביולוגיות מורכבות

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

חזותי יחיד מולקולת הדנ&quot;א שכפול עם מיקרוסקופ פלואורסצנטי

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

סינתזה כיול nanoprobes זרחני עבור הדמיה חמצן במערכות ביולוגיות

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Lineage תיוג של תאים דג הזברה עם לייזר Uncagable והעמסת dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

שיטה פשוטה הדמיה ארבידופסיס מותיר שימוש Perfluorodecalin כנושאת הדמיה Infiltrative

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

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 ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering הומולוגי רקומבינציה בבונה<em&gt; דרוזופילה</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...