نشكر عايدة دي لا كروز لتطوير وتدريس البروتوكول الأصلي الذي يستند هذا الإصدار 10. ويدعم عمل في مختبر من قبل المعاهد الوطنية للصحة Buttitta منحة GM086517.

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الكتاب ليس لديهم ما يكشف.

بروتوكول الموصوفة هنا يسمح لتحليل دورة الخلية، حجم الخلية النسبي وعدد الخلايا في الأنسجة الحية النسبية ذبابة الفاكهة في مراحل النمو المختلفة. عندما يقترن هذا التحليل مع نوع من الخلايا المحددة التعبير بروتين فلوري أو النسب تتبع، ويمكن الحصول على معلومات مفصلة ?...

التدفق الخلوي يمكن أن تستخدم لقياس بقاء الخلية، حجم الخلية النسبي، والمحتوى الحمض النووي والتعبير بروتين فلوري في السكان الخلية الحية. ونظرا لتكرار الحمض النووي النووية خلال S-المرحلة، معلومات عن محتوى الحمض النووي في السكان من الخلايا يمكن استخدامها لاستنتاج النسب المئوية النسبية في مختلف مراحل دورة الخلية 1-3. وقد أصبح هذا الأسلوب حجر الزاوية في تحليل دورة الخلية في نظم نموذج من الخميرة إلى الثدييات. أصبح ذبابة الفاكهة ذبابة الفاكهة السوداء البطن نظام نموذجا ممتازا لالوراثية في التحليلات المجراة من تنظيم دورة الخلية. الأدوات الجينية الواسعة المتاحة في الذباب تسمح للتلاعب الأنسجة أنيقة محددة زمنيا وينظم من المنظمين دورة الخلية في الجسم الحي جنبا إلى جنب مع الفلورسنت نسب البروتين على أساس تتبع 4-6. وقد استخدم التدفق الخلوي لدراسة محتوى الحمض النووي في عدد من أنواع الخلايا ذبابة الفاكهة، بما في ذلك endoreplicating الخلايا والخلايا الإنقسامية مثقف 7،8. وقدم تقدما هاما للدراسات في دورة الخلية الحية من قبل دي لا كروز وادغار، مع وضع بروتوكول لتحليل تدفق cytometric من العيش مضاعفا ذبابة الفاكهة أقراص تخيلي 9،10، وهو البروتوكول الذي تم استخدامها وتكييفها من قبل العديد من مختبرات. هذه التقنية، عندما يقترن الوراثية في الجسم الحي تتبع النسب عن طريق محرض التعبير بروتين فلوري ووضع العلامات المحددة الأنسجة، ويسمح احد للحصول على معلومات حول آثار التلاعب الجيني في العام الخلية مضاعفة الوقت، وحجم الخلية وتحديد التوقيت الدقيق لمراحل دورة الخلية في الجسم الحي 9 ، 11. ومع ذلك فقد اعتمدت هذه الطريقة حتى الآن على استخدام الخلية نفاذية هويشت 33342 صبغ الحمض النووي الإقحام إلى وصمة عار وتحديد الحمض النووي في الخلايا الحية، والتي حدت مستخدمين تتدفق cytometers مع ليزر الأشعة فوق البنفسجية قادرة على إثارة صبغ هويشت. وتوجد عموما هذه فقط في مطابقة (أي دينار بحريني FACS VANTAGE، BD FACSAria) أو مكلفة متعدد الألوان أنظمة الفوق (أي BD LSR)، والتي تتطلب عادة دعم المرافق المؤسسية الأساسية التدفق. قمنا بتعديل بروتوكول المستندة إلى هويشت لاستخدام صبغة DNA الخلية الحية الجديدة من إينفيتروجن، Vybrant DyeCycle البنفسجي. هذه الصبغة هي متوافقة مع البنفسجي 405 نانومتر ليزر، أكثر شيوعا في تحليل الفوق أصغر والمتاحة في الصغيرة محلل الفوق مكتفية ذاتيا، والصوتية أقلم التركيز عداد الكريات. هنا نقدم بروتوكول مفصلة لتحليل دورة الخلية التي يمكن أن يقترن مع نوع من الخلايا، حجم الخلية، عدد الخلايا وتحليل النسب في مجموعة متنوعة من الأنسجة ذبابة الفاكهة خلال مراحل مختلفة من التطوير باستخدام DyeCycle البنفسج وأقلم. هذا البروتوكول يوسع عدد من cytometers مناسبة لمثل هذا التحليل مع الأنسجة ذبابة الفاكهة ويقدم أمثلة عن كيفية هذا النوع من تحليل دورة الخلية الحية يمكن تعديلها لأنواع الأنسجة الإضافية ومراحل النمو.

<table><tbody><tr><td>12x75 mm Polystyrene Round-Bottom 5 ml Test Tube </td><td>BD Falcon</td><td>352058</td><td>5 ml tubes</td></tr><tr><td>Attune Acoustic Focusing Cytometer</td><td>Life Technologies/ Applied Biosystems</td><td>4445315</td><td>Blue / Violet configuration</td></tr><tr><td>Attune Cytometer Software (version 1.2.5)</td><td>Life Technologies/ Applied Biosystems</td><td>&lt;strong&gt;&lt;em&gt;Free&lt;/em&gt;&lt;/strong&gt;</td><td>PC only</td></tr><tr><td>Attune Performance Tracking Beads (5 x 106 beads/ ml) </td><td>Life Technologies/ Applied Biosystems</td><td>4449754</td><td>For daily performance test</td></tr><tr><td>Dumont #5 Inox forceps </td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Embryo dishes 30 mm x 12mm </td><td>Electron Microscopy Sciences</td><td>70543-30</td><td>Glass dissection dishes </td></tr><tr><td>Eppendorf Thermomixer</td><td>Eppendorf</td><td>022670051</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma</td><td>T4174</td><td></td></tr><tr><td>Vannas-T&amp;#252;bingen Spring Scissors </td><td>Fine Science Tools</td><td>15003-08</td><td>Straight 5mm Cutting Edge </td></tr><tr><td>Vybrant DyeCycle Violet Stain</td><td>Life Technologies/ Invitrogen</td><td>V35003</td><td></td></tr><tr><td colspan="4">&lt;span class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table 1. Required reagents and instruments.&lt;/strong&gt;&lt;/span&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Live DNA Stain Solution (10 ml):&lt;/p&gt; &lt;p class=&quot;jove_content&quot;&gt;1 ml 10X Ca&lt;sup&gt;2+&lt;/sup&gt; Mg&lt;sup&gt;2+&lt;/sup&gt; free PBS (pH7.2)&lt;br/&gt; 9 ml 10X Trypsin-EDTA (Sigma)&lt;br/&gt; 5 &amp;mu;l Invitrogen Vybrant DyeCycle Violet (note that this is 0.25X the recommended concentration for mammalian cells. We find that higher concentrations are toxic to &lt;em&gt;Drosophila&lt;/em&gt; cells.)&lt;/p&gt; &lt;p class=&quot;jove_content&quot;&gt;10X Ca&lt;sup&gt;2+&lt;/sup&gt; Mg&lt;sup&gt;2+&lt;/sup&gt; free PBS (pH7.2): 1.37M NaCl, 27 mM KCl, 100mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (dibasic), 20mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (monobasic) adjusted to pH 7.2&lt;/p&gt; </td></tr><tr><td colspan="4">&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt; &lt;/td&gt; &lt;td&gt;&lt;strong&gt;FSC&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;SSC&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;BL1&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;VL1&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Threshold (x1000)&lt;/td&gt; &lt;td&gt;100&lt;/td&gt; &lt;td&gt;10&lt;/td&gt; &lt;td&gt;10&lt;/td&gt; &lt;td&gt;10&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Voltage (mV)&lt;/td&gt; &lt;td&gt;2950&lt;/td&gt; &lt;td&gt;4250&lt;/td&gt; &lt;td&gt;1800&lt;/td&gt; &lt;td&gt;1150&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt; &lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table 2. Threshold and voltage setting for the analysis in Figure 2.&lt;/strong&gt;&lt;/p&gt;</td></tr></tbody></table>

live cell cycle analysis of drosophila tissues using the attune acoustic focusing cytometer and vybrant dyecycle violet dna stain

وقد التدفق الخلوي تستخدم على نطاق واسع للحصول على معلومات حول محتوى الحمض النووي في عدد السكان من الخلايا، إلى استنتاج النسب المئوية النسبية في مختلف مراحل دورة الخلية. وقد تم تمديد هذه التقنية بنجاح إلى الأنسجة الإنقسامية من طراز كائن ذبابة الفاكهة السوداء البطن للدراسات الجينية من تنظيم دورة الخلية في الجسم الحي. عندما يقترن مع نوع من الخلايا المحددة التعبير بروتين فلوري والتلاعب الجيني، يمكن للمرء الحصول على معلومات مفصلة حول الآثار المترتبة على عدد الخلايا، حجم الخلية ودورة الخلية الإلغاء في الجسم الحي. ومع ذلك فقد اعتمدت هذه الطريقة الخلية الحية على استخدام الخلية نفاذية هويشت 33342 صبغ الحمض النووي الإقحام، مما يحد من المستخدمين إلى أجهزة قياس التدفق الخلوي مجهزة ليزر الأشعة فوق البنفسجية. قمنا بتعديل هذا البروتوكول على استخدام الخلية الحية صبغ الحمض النووي أحدث وVybrant DyeCycle البنفسج، متوافقة مع البنفسج الليزر 405nm وأكثر شيوعا. بروتوكول المعروضة هنا يسمح لكفاءة تحليل دورة الخلية إلى جانب الخليةنوع، حجم الخلية النسبي والمعلومات عدد الخلايا، في مجموعة متنوعة من الأنسجة ذبابة الفاكهة. هذا البروتوكول تمتد دورة الخلية تقنية مفيدة لتحليل الأنسجة الحية ذبابة الفاكهة إلى محلل الفوق الصغيرة، والصوتية أقلم التركيز عداد الكريات، والتي يمكن تشغيلها وصيانتها على نطاق و-LAB واحد.

ويبين الشكل 2 نتائج ممثلة لعينة الجناح اليرقات، معربا عن GFP في النصف الخلفي من الأنسجة، وذلك باستخدام قالب GFP المقدمة. ويتم الحصول على نتائج مماثلة مع نوع الأنسجة نفسها، ونمط التعبير عن طلب تقديم العروض باستخدام طلب تقديم العروض قالب المقدمة (الشكل 3A).</stro...

Watch this Scientific Journal Video about Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain at JoVE.com

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

بروتوكول للتحليل دورة الخلية الحية<em&gt; ذبابة الفاكهة</emيوصف&gt; الأنسجة باستخدام الصوتية أقلم التركيز عداد الكريات. هذا البروتوكول يوفر في الوقت نفسه المعلومات حول حجم الخلية النسبي، عدد الخلايا، والمحتوى الحمض النووي ونوع من الخلايا عن طريق تتبع نسب أو الأنسجة التعبير محددة من البروتينات الفلورية<em&gt; في الجسم الحي</em&gt;.

تحليل دورة الخلية الحية من<em&gt; ذبابة الفاكهة</em&gt; الأنسجة باستخدام الصوتية أقلم التركيز عداد الكريات وVybrant DyeCycle البنفسج الحمض النووي وصمة عار

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

live-cell-cycle-analysis-drosophila-tissues-using-attune-acoustic

Research

JoVE Journal

Biology

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

13.6K Views.  University of Michigan. بروتوكول للتحليل دورة الخلية الحية ذبابة الفاكهة الأنسجة باستخدام الصوتية أقلم التركيز عداد الكريات. هذا البروتوكول يوفر في الوقت نفسه المعلومات حول حجم الخلية النسبي، عدد الخلايا، والمحتوى الحمض النووي ونوع من الخلايا عن طريق تتبع نسب أو الأنسجة...

Video: Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

استخدام خلايا Drosophila S2 للتصوير الحي لقسم الخلايا

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

Genetics

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.

تحليل ديناميات Actomyosin في جداول الخلوية والأنسجة المحلية باستخدام أفلام الفاصل الزمني من غرف البيض Drosophila المستزرعة

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

Developmental Biology

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

التصوير والتحليل لاتجاهات الأنسجة وديناميات النمو في الظهارة الهوائية النامية Drosophila خلال مراحل البوبال

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

إعداد دروسوفيلا رفال واختبارات Pupal لتحليل انقسام الخلايا في الأنسجة الحية والسليمة

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

تحليل المركز دورة الخلية في خلايا الثدييات

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

دراسة حاجز الميتوزى بواسطة يوضح الحيوي Kinetochore السلوك البروتين والحركة كروموسوم في المعيشة<em&gt; ذبابة الفاكهة</em&gt; الجنين المخلوي

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

تحليل الخلوي الحيوانات المنوية: الأعمال التحضيرية لايف والثابتة من<em&gt; ذبابة الفاكهة</em&gt; الخصية

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

التصوير من خلال حالة العذراء من<em&gt; ذبابة الفاكهة السوداء البطن</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S&gt;G2/M (progression from S to G2/M), and M phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

5-إيثينيل-2'-Deoxyuridine/فوسفو-هيستون H3 بروتوكول وضع العلامات المزدوجة لتحليل تطور دورة الخلية في الخلايا الجذعية العصبية Drosophila

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) ...

An Introduction to Cell Division

Cell division is the process by which a parent cell divides and gives rise to two or more daughter cells. It is a means of reproduction for single-cell organisms. In multicellular organisms, cell division contributes to growth, development, repair, and the generation of reproductive cells (sperms and eggs). Cell division is a tightly regulated process, and aberrant cell division can cause diseases, notably cancer. 
JoVE's Introduction to Cell Division will cover a brief history of the landmark discoveries in the field. We then discuss several key questions and methods, such as cell cycle analysis and live cell imaging. Finally, we showcase some current applications of these techniques in cell division research.

Cell Division, Proliferation, Cancer and Cell Cycle Analysis

Cell division is the process by which a parent cell divides and gives rise to two or more daughter cells. It is a means of reproduction for single-cell ...

تحليل دورة الخلية الحية من

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