This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), Takeda Science Foundation (HT), and MEXT-Supported Program for the Strategic Research Foundation at Private Universities (HT).

הערה: הזבובים המהונדסים נעשו שימוש במחקר זה כולל את הפעולות הבאות: DE-CAD :: GFP 10. 1. הכנת הצלחת האפלה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכינו תערובת של אגר 12.5 גרם, 125 מ&quot;ל 100% מיץ תפוחים זמינים מסחרית, 12.5 גלוקוז g, ו- 375 מ&quot;ל H 2 O. מחממים את התערובת במיקרוגל ויוצקים אותו לתוך צלחת תרבית תאים 3 ס&quot;מ. אחסן את התערובת על 4 מעלות צלזיוס לשימוש עתידי. </li><li style=";text-align:right;direction:rtl"> לאחר הכנת צלחת התפוח, להוסיף שכבה דקה של ממרח שמרים לש ​​על גבי זה כדי לאפשר הזבובים להטיל ביצים. </li></ol> 2. הכנת עובר ופרוטוקול הדמיה חיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים כ 50 זבובים (25 זכרים ו -25 נקבות) בבקבוק להזדווג ובהמשך להטיל ביצים לילה על צלחת תפוח מכוסה שמרים לשה. הגדר את צלחת מיץ תפוחים בפתחו של הבקבוק. שמור את הבקבוק באינקובטור. עוטפים את בקבוקי פלסטיק תסיסנית המניות ברדיד אלומיניום כדי להנחות הדואר טס להטיל יותר ביצים. </li><li style=";text-align:right;direction:rtl"> למחרת בבוקר, להחליף את צלחת מיץ תפוחים הישן בחדש. </li><li style=";text-align:right;direction:rtl"> בואו הזבובים להטיל ביצים במשך 3 עד 4 שעות על ידי לפפה את הבקבוק בנייר אלומיניום. </li><li style=";text-align:right;direction:rtl"> שלוש עד ארבע שעות מאוחר יותר, לאסוף את העוברים בתוך מסננת העובר מהצלחת מיץ תפוחים באמצעות רע מברשת או כותנה ולשטוף אותם עם PBS. לשטוף את העוברים 2 עד 3 פעמים יותר עם PBS במנות תרבות 12 גם. </li><li style=";text-align:right;direction:rtl"> Dechorionate את העוברים עם אקונומיקה 50% במשך 5 דקות במנות תרבות 12 גם. </li><li style=";text-align:right;direction:rtl"> בעקבות תהליך dechorionation, לשטוף את העוברים 2 עד 3 פעמים יותר עם PBS. </li><li style=";text-align:right;direction:rtl"> מעבירים את העוברים על צלחת תרבית תאים 3 ס&quot;מ המכיל הבמה PBS ובחר 5 עוברים תחת המיקרוסקופ מבוסס על רמת שקיפות גבולותיהן. פיפטה עובר המבוים באמצעות קצה פיפטה 200 μL. </li><li style=";text-align:right;direction:rtl"> בשלב הבא, במקום שני עוברים שנבחרו על coverslip זכוכית, ולהסיר PBS העודף בנייר טישו מעוות דק. אוריינט עובר בצד הגבה עלאת coverslip באמצעות נייר טישו מעוות דק. לאחר מכן, לצרף את עוברי coverslip הזכוכית עם גריז סיליקון באמצעות מחט דקה ונייר דק מעווה. </li><li style=";text-align:right;direction:rtl"> הוסף טיפה קטנה של שמן halocarbon 700 על העוברים ומניחים את coverslip המכיל את העוברים במהופך בשקופית מסוכסך, משאיר קצת מקום בתחתית המגלשה. </li><li style=";text-align:right;direction:rtl"> בדוק את העוברים באמצעות מערכת הדמיה confocal, לייזר ארגון / 488, אובייקטיבי טבילה-שמן (63X). הערה: זיהוי העובר בשלב ההתפתחותי המתאים חשובה ללכוד תמונות של אירוע gastrulation. בתנאים אלה, יש למלא אחר כמעט כל העוברים להתפתח בצורה תקינה עד לשלב לפחות 14. העובר נתח יכול להיות מתורבת להמשיך לפתח כמבוגר. </li></ol>

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The authors have nothing to disclose.

Although we have previously reported a similar procedure to capture live images of the gastrulation process in Drosophilla1, the method we describe here is detailed and easy to trace endogenous cadherin expression and thus is quite useful for genetic screening of key factors involved in gastrulation. To maximize success with this imaging procedure, it is essential to use an indented slide. Mechanical pressure sometimes causes embryonic death. Therefore, it is also important to handle the embryos as ge...

Gastrulation הוא הסט הראשון של אירועים דינמי מורפולוגית המתרחשים במהלך ההתפתחות העוברית של בעלי חיים רב-תאיים כגון תסיסנית 1,2. מעניין, המתעוררים הראיות עולה כי תהליך זה מוסדר באמצעות משחק הגומלין בין מנגנונים מכניים ומולקולרית 3. יתר על כן, אפיתל המעבר mesenchymal (EMT), שהוא תהליך מכריע gastrulation, הוא מעורב גם בתהליכי מחלות אנושיות כגון גרורות סרטן 4-8. כמו גנים כאלה, רבים השולטים התכווצות פסגה גם ידועים כגורמי מפתח EMT שנצפתה בסרטן גרור 9. לפיכך, מועקה הפסגה בעת gastrulation הוא מודל מצוין לחקור את מנגנוני ויסות הנ&quot;ל כדי לשפר את ההבנה שלנו של גרורות סרטניות. היתרון של שיטה זו הוא כי אנו יכולים לצפות תנועת התא בעת gastrulation בזמן אמת ולכן, אנחנו נהיהמסוגל להקרין גנים המעורבים gastrulation וכן גרורות סרטניות. למרות יחסית ידוע, תא אל התא הדבק הוא חשב לשחק תפקיד מרכזי התכווצות פסגת 1. גנטיקת תסיסנית מתאימה גם לחקירות ברמת התא בודדות לחקור מנגנונים מולקולריים רגולטוריות. מודל זה יאפשר לנו לחשוף את החשיבות של התכווצות הפסגה במהלך gastrulation. יתר על כן, שיטה זו יכולה לשמש מסך גנים המעורבים בהתפשטות גרורות סרטניות. תמונות חיות לכידות של gastrulation תסיסנית נוספת אפשרו לנו להבין ביתר פירוט את המנגנונים המולקולריים השולטים התארגנות רקמות. בזאת, אנו מספקים תיאור מקיף של שיטה פשוטה כדי להשיג זאת.

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			<td height="54" style="height:54px;width:268px;">Halocarbon oil 700</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:129px;">MKBH 5726</td>
			<td style="width:133px;"></td>
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		<tr height="54">
			<td height="54" style="height:54px;width:268px;">Vacuum grease Silicone</td>
			<td style="width:164px;">Beckman</td>
			<td style="width:129px;">335148</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Glass coverslip&#160;</td>
			<td style="width:164px;">Matsunami glass</td>
			<td style="width:129px;">Thickness No1</td>
			<td style="width:133px;">24-36mm</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Embryo stariner</td>
			<td style="width:164px;">Corning</td>
			<td style="width:129px;">Corning3477</td>
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			<td height="38" style="height:38px;width:268px;">Plastic Drosophilla Stock Bottles</td>
			<td style="width:164px;">Hitec</td>
			<td style="width:129px;">MKC-100</td>
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			<td height="21" style="height:21px;width:268px;">DE-Cadherin knock-in flies</td>
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			<td>REF (10)</td>
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imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies

Gastrulation הוא הסט הראשון של אירועים דינמיים מורפולוגית המתרחשים במהלך ההתפתחות העוברית של בעלי חיים רבים-תאיים כגון תסיסנית. שינוי מורפולוגי זו מוכרת גם בשם אפיתל המעבר mesenchymal (EMT). חוסר ויסות של EMT קשורה סיסטיק ו גרורות סרטניות. יש ראיות המתעוררים כי EMT נשלטת על ידי מספר מנגנונים מולקולריים. כמו גנים מפתח כגון, רבים השולטים התכווצות הפסגה גם ידועים להיות מרכיב חשוב EMT ציין בהתפשטות גרורות סרטניות. כמו EMT במהלך gastrulation תסיסנית, תאי אפיתל יכולים להיגרם לשנות את צורתם ואת לתכנת מחדש להפנות תא גורל כלפי סוגי תאים שונים אחרים. כאן אנו מספקים שיטת הדמיה חזקה של gastrulation תסיסנית כדי assay את החניכה של תנועות הסלולר המוךפו&quot;גנטי שהולכות וזיהוי תאי גורל בשלב זה של התפתחות עוברית. באמצעות שיטה זו, אנו מזהים גell התארגנות בזמן gastrulation ולהפגין את החשיבות של התכווצות הפסגה במהלך gastrulation באמצעות GFP שכותרתו DE-cadherin.

כאן, אנו מציגים את אירועי gastrulation של עובר תסיסנית סקירה כללית של הליך הכנת עובר (איור 1). קרום התא מסומנים באמצעות DE-cadherin :: GFP ו הדמיה חיה של תנועות תא מתבצע בעת gastrulation תסיסנית (איור 2). מאז זבובי DE-cadherin GFP מאפשרים לנו לדמיין צמתים דב...

Watch this Scientific Journal Video about Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies at JoVE.com

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

הדמיה של שינוי צורה של התא ותנועתו ב<em&gt; תסיסנית</em&gt; Gastrulation שימוש Reporter DE-cadherin זבובים טרנסגניים

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

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Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

7.0K Views.  Jahangirnagar University. Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

Video: Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

מעקב תאים דג הזברה GFP מהונדס שימוש במערכת photoconvertible PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

ניתוח<em&gt; In vivo</em&gt; נדידת תאים באמצעות השתלות תא והדמיה זמן לשגות עוברי דג הזברה

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

תרבות של מבוגר דג הזברה טראנסגנטי רשתית Explants עבור Live- תא הדמיה ידי multiphoton מיקרוסקופי

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

הדמיה חיה של עכבר משניים פיוז &#39;ן

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

חיתוך, צביעה של השחלות הגולמי דרוזופילה

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

וזמינותו מבוססת תא לחקור שרירים שאינם צולבות הקישור חוטים שרירן II Contractility ויה gastrulation מקופל איתות לשביל בתאים S2R + דרוזופילה

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

ניתוח של Actomyosin בסולמות הסלולר והרקמה המקומית באמצעות הזמן- סרטים פקיעה של מתורבת ביצת ביצה צ'יימברס

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

מפשיר Culturing, Cryopreserving שורות תאים דרוזופילה

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

הדמיה וניתוח עורקי קשת והלוע באמצעות שחזור-הר אימונוהיסטוכימיה ותלת מימד

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

הדמיה מוטות Actin תוך-גרעיניים בחום חי הדגישו עוברי דרוזופילה

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...