GFP-β-אקטין פלסמיד סופק בנדיבות על ידי ד&quot;ר ווין אור, LSUHSC-S המחלקה לפתולוגיה, והיה מוגבר במעבדה של ד&quot;ר בקי Worthylake, LSUHSC-NO המחלקה לפרמקולוגיה. עבודה זו נתמכה על ידי מענקים מהמוסד הלאומי לבריאות (P20 RR-018766) ואת איגוד הלב האמריקני (05835386N).

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אין ניגודי אינטרסים הכריז.

הדמיה של ה-GFP-אקטין בתאי אנדותל לחיות מאפשר ניתוח מפורט של הדינמיקה של cytoskeleton אקטין בתגובה לגירויים דלקתיים. שיטה זו עשויה להיות שימושית גם לבנות על ממצאים קודמים מראה שיפוץ cytoskeleton בתגובה כוחות פיזיים כמו מתח גזירה 11. בנוסף, שיטה זו מאפשרת הערכה מפורטת של התרומה של הדינמיקה אקטין cytoskeletal על פעילויות שונות תא האנדותל, כולל הגירה, מיטוזה, היווצרות של צמתים אינטר והתבגרות junctional, תפקוד ותחזוקה של המכשול. ב הנתונים המוצגים, התנהגותם של cytoskeleton אקטין אנדותל ניתן לראות לפני ואחרי הטיפול עם תרומבין. Lamellipodia מקומי כל בשולי לתאי אנדותל נצפו להרכיב רגרסיה לאורך זמן monolayers התא הן nonconfluent ומחוברות. טיפול עם תרומבין בקצרה קטע טופס lamellipodiaation מחזור. טרומבין גם גרמה לתאים להתכווץ מעט, בהסכמה עם דיווחים קודמים כי תרומבין גורמת ללחץ סיבי אקטין היווצרות מתח מוגבר להתפתחות צנטריפטלי בתאי אנדותל 12-14. עם זאת, מן המחקרים תא חי הדמיה כגון זה, מקורם של הסיבים הלחץ כעת ניתן נחוש. ב HUVEC, רוב הסיבים מתח מקורן בשולי התא דומה סיבי קשת רוחבי של תאים נודדים 8, 9. כוח נוסף של שיטה זו על פני שימוש בתאים קבועים שמספר סיבי המתח ניתן לכמת בתאים בודדים לפני ואחרי הטיפול תרומבין, ביטול הטיית בחירה בין קבוצות הניסוי. באמצעות פרוטוקול זה אנו מעריכים תנועה דינמית של קצה תא הלחץ סיבי אקטין. כדי להבין את הדינמיקה מונומר אקטין בתאי אנדותל, טכניקות מתקדמות יותר כגון התאוששות הקרינה לאחר photobleaching (FRAP) או פלואורסצנטי רבב מיקרועותק (FSM) יכול להיות מיושם 15, 16. בנוסף, בגלל כלי הדם לתאי אנדותל יכול לייצג מודל טוב יותר של תפקוד מחסום כלי הדם, אופטימיזציה של פרוטוקולים transfection לבטא בצורה יעילה GFP-אקטין בתאי אנדותל כלי הדם מייצג הכיוון העתידי הגיוני. לסיכום, הדמיה של בתאי האנדותל לחיות להביע GFP-אקטין מהווה כלי רב עוצמה כדי לקבוע כיצד cytoskeleton אקטין תא האנדותל מגיב סוגים שונים של גירויים. מחקרים באמצעות monolayers אנדותל ומחוברות היטב תסייע לקבוע את התפקידים של מבנים דינמיים כגון אקטין עשיר lamellipodia ו רוחבי מתח קשת סיבים בתפקוד האנדותל מחסום. בנוסף, הדמיה של תא חי לתאי אנדותל להביע GFP-אקטין או חלבונים היתוך אחרים המאפשרים הדמיה של מבנים subcellular אחרים יספק מידע spatiotemporal מפורט כדי להבין את מנגנוני איתות מבניים דetermine שלמות המכשול.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="4"> 1. רינגר 5x במלאי </td></tr><tr><td> כימי </td><td> חברה </td><td> מספר קטלוג </td><td> כמות </td></tr><tr><td> נתרן כלוריד </td><td> EMD </td><td> SX0420-3 </td><td> 35 גרם </td></tr><tr><td> אשלגן כלוריד </td><td> JT בייקר </td><td> 3040 </td><td> 1.75 גר &#39; </td></tr><tr><td> סידן כלוריד </td><td> סיגמא </td><td> C-3881 </td><td> 1.47 גר &#39; </td></tr><tr><td> מגנזיום סולפט </td><td> סיגמא </td><td> M-9397 </td><td> 1.44 גר &#39; </td></tr><tr><td> מים מסוננים ועיקור </td><td> N / A </td><td> N / A </td><td> מביאים L 1 </td></tr><tr><td colspan="4"> סינון סטרילי לתוך בקבוקים חנויות autoclaved ב 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2. מגבים חיץ </td></tr><tr><td> כימי </td><td>חברה </td><td> מספר קטלוג </td><td> כמות </td></tr><tr><td> מגבים </td><td> סיגמא </td><td> M3183 </td><td> 125.6 גרם </td></tr><tr><td> מים מסוננים ועיקור </td><td> N / A </td><td> N / A </td><td> מביאים L 1 </td></tr><tr><td colspan="4"> סינון סטרילי לתוך בקבוקים חנויות autoclaved ב 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3. הפתרון אלבומין מלח פיזיולוגיים (APSS) </td></tr><tr><td> כימי </td><td> חברה </td><td> מספר קטלוג </td><td> כמות </td></tr><tr><td> רינגר מניות (5x) </td><td> N / A </td><td> N / A </td><td> 200 מ&quot;ל </td></tr><tr><td> מגבים הצפת </td><td> N / A </td><td> N / A </td><td> 5 מ&quot;ל </td></tr><tr><td> סודיום פוספט </td><td> סיגמא </td><td> S-9638 </td><td> 0.168 גרם </td></tr><tr><td> Pyruvate נתרן &lt;/ Td&gt; <td> סיגמא </td><td> P5280 </td><td> 0.22 גר &#39; </td></tr><tr><td> מלח נתרן EDTA </td><td> סיגמא </td><td> ED2SS </td><td> 0.0074 גרם </td></tr><tr><td> גלוקוז </td><td> סיגמא </td><td> G7528 </td><td> 0.901 גרם </td></tr><tr><td> אלבומין, שור </td><td> USB </td><td> 10856 </td><td> 10 גרם </td></tr><tr><td> מים מסוננים ועיקור </td><td> N / A </td><td> N / A </td><td> מביאים L 1 </td></tr><tr><td colspan="4"> התאם את ה-pH 7.4 בשעה 37 ° C, ואז לסנן לתוך בקבוקים סטריליים autoclaved ולאחסן ב 4 ° C. </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4. 0.9% Saline </td></tr><tr><td> כימי </td><td> חברה </td><td> מספר קטלוג </td><td> כמות </td></tr><tr><td> נתרן כלוריד </td><td> EMD </td><td> SX0420-3 </td><td> 9 גרם </td></tr><tr><td> מים מסוננים ועיקור </td><td> N / A </td><td> N / A </td><td> מביאים L 1 </td></tr><tr><td colspan="4"> סינון סטרילי לתוך בקבוקים חנויות autoclaved ב 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5. הפתרון לטין 1.5% </td></tr><tr><td> לטין מהעור חזירי </td><td> סיגמא </td><td> G2500 </td><td> 15 גרם </td></tr><tr><td> 0.9% Saline </td><td> N / A </td><td> N / A </td><td> מביאים L 1 </td></tr><tr><td colspan="4"> חם הפתרון 37 ° C כדי להמיס הג&#39;לטין מספיק. אמנם עדיין חמים, לסנן לתוך בקבוקים סטריליים autoclaved ולאחסן ב 4 ° C </td></tr></tbody></table>

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

האנדותל כלי הדם ממלא תפקיד חשוב כמחסום סלקטיבי חדיר נוזלים מומסים. צמתים דבק בין לתאי אנדותל להסדיר החדירות של האנדותל, ומחקרים רבים הראו את תרומתו החשובה של cytoskeleton אקטין לקביעת שלמות junctional 1-5. חגורת אקטין קורטיקלי נחשב חשוב לשמירה על יציבות צמתים 1, 2, 4, 5. לעומת זאת, סיבי אקטין מתח נחשבים ליצור מתיחות בתוך צנטריפטלי ותאי האנדותל כי מחלישה צמתים 2-5. הרבה תיאוריה זו התבססה על מחקרים שבהם לתאי אנדותל מטופלים עם מתווכים דלקתיים ידוע להגדיל חדירות האנדותל, ולאחר מכן תיקנה את התאים תיוג F-אקטין להסתכלות מיקרוסקופית. עם זאת, מחקרים אלו מספקים הבנה מוגבלת מאוד של תפקיד cytoskeleton אקטין כי תמונות של תאים קבוע לספק תמונות בלבדזמן ללא מידע על הדינמיקה של מבנים אקטין 5. Live-תא הדמיה מאפשר שילוב של האופי הדינמי של cytoskeleton אקטין לתוך המחקרים של מנגנוני קביעת שלמות מחסום אנדותל. היתרון העיקרי של שיטה זו הוא כי ההשפעה של גירויים דלקתיים שונים על מבנים אקטין בתאי אנדותל ניתן להעריך אותה קבוצה של תאים חיים לפני ואחרי טיפול, הסרת הטיה פוטנציאלית שעלולה להתרחש בעת התבוננות דגימות קבועות. האדם וריד הטבור לתאי אנדותל (HUVEC) הם transfected עם אקטין GFP-β פלסמיד וגדל מפגש על coverslips זכוכית. זמן לשגות תמונות של ה-GFP-אקטין ב HUVEC ומחוברות נלכדים לפני ואחרי תוספת של מתווכים דלקתיים כי לעורר תלוי זמן שינויים שלמות מחסום אנדותל. מחקרים אלה מאפשרים תצפית ויזואלית של רצף נוזל שינויים cytoskeleton אקטין שתורמים endotheliaאני שיבוש מחסום ושיקום. התוצאות שלנו מראים בעקביות המקומי, אקטין עשיר היווצרות lamellipodia מחזור בתאי אנדותל. התהוות ותנועה של סיבי אקטין הלחץ יכול להיות גם ציין. ניתוח תדירות היווצרות מחזור של lamellipodia המקומית, לפני ואחרי הטיפול עם גירוי דלקתי יכול להיות מתועד על ידי מנתח רשם גלים. מחקרים אלו מספקים מידע חשוב על אופייה הדינאמי של cytoskeleton אקטין בתאי אנדותל שניתן להשתמש כדי לגלות המנגנונים המולקולריים מזוהה קודם לכן חשוב לשמירה על שלמות המחסום אנדותל.

Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

הדמיה מיקרוסקופית של תאים אנדותל לחיות להביע GFP-אקטין מאפשר אפיון של שינויים דינמיים מבנים cytoskeletal. שלא כמו טכניקות להשתמש דגימות קבוע, שיטה זו מספקת הערכה מפורטת של השינויים הזמני cytoskeleton אקטין בתאי אותו לפני, במהלך ואחרי גירויים פיזיים, תרופתי, או דלקתיים שונים.

עיון cytoskeleton אקטין ב לתאי אנדותל חיים הבעת GFP-אקטין

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

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The Cytoskeleton I: Actin and Microfilaments

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27.1K Views.  Louisiana State University Health Sciences Center. הדמיה מיקרוסקופית של תאים אנדותל לחיות להביע GFP-אקטין מאפשר אפיון של שינויים דינמיים מבנים cytoskeletal. שלא כמו טכניקות להשתמש דגימות קבוע, שיטה זו מספקת הערכה מפורטת של השינויים הזמני cytoskeleton אקטין בתאי אותו לפני, במהלך ואחרי גירויים פיזיים, תרופתי, או דלקתיים שונים.

Video: Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

בדיקת רגישות חזותית על מהירות ועל כיוון התנועה של לטאות

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

דימות של תא חי F-אקטין Dynamics באמצעות פלורסנט מיקרוסקופית רבב (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

הדמיה חיה של תנועתיות התא ואת cytoskeleton אקטין של נוירונים בודדים לבין תאים קרסט עצבית בעוברים דג הזברה

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

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

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

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

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

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

חי הדמיה של חיסול תאים אפופטוטיים במהלך<em&gt; דרוזופילה</em&gt; עובר

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

מיקרוסקופית Intravital למבני ההדמיה subcellular בעכברים חיים לבטא חלבוני פלורסנט

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Assay היווצרות צינור תא האנדותל ל<em&gt; במבחנה</em&gt; חקר אנגיוגנזה

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

ניתוח של סטרס חמצוני בעוברי דג הזברה

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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 ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

זמן לשגות הדמיה ברזולוציה גבוהה וניתוח אוטומטי של Dynamics microtubule In Living האדם טבורי וריד לתאי אנדותל

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...