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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+BrdU+labeling+and+nuclear+flow+sorting+of+the+Drosophila+ovary.">Fluorescent BrdU labeling and nuclear flow sorting of the Drosophila ovary.</a> Methods Mol. Biol. 247, 203-213 (2004).</li><li>Bjorklund, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pathways+regulating+cell+size+and+cell-cycle+progression+by+RNAi.">Identification of pathways regulating cell size and cell-cycle progression by RNAi.</a> Nature. 439, 1009-1013 (2006).</li><li>Neufeld, T. P., de la Cruz, A. F., Johnston, L. A., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+growth+and+cell+division+in+the+Drosophila+wing.">Coordination of growth and cell division in the Drosophila wing.</a> Cell. 93, 1183-1193 (1998).</li><li>de la Cruz, A. F., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+Drosophila+cells.">Flow cytometric analysis of Drosophila cells.</a> Methods Mol. Biol. 420, 373-389 (2008).</li><li>Reis, T., Edgar, B. 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A. 93, 11687-11692 (1996).</li><li>Schubiger, M., Palka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+spatial+patterns+of+DNA+replication+in+the+developing+wing+of+Drosophila.">Changing spatial patterns of DNA replication in the developing wing of Drosophila.</a> Dev. Biol. 123, 145-153 (1987).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila; A laboratory handbook. , (1989).</li><li>Theodosiou, N. A., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+FLP/FRT+system+to+study+Drosophila+development.">Use of FLP/FRT system to study Drosophila development.</a> Methods. 14, 355-365 (1998).</li><li>Freeman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reiterative+use+of+the+EGF+receptor+triggers+differentiation+of+all+cell+types+in+the+Drosophila+eye.">Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye.</a> Cell. 87, 651-660 (1996).</li><li>Su, T. T., Sprenger, F., DiGregorio, P. J., Campbell, S. D., O'Farrell, P. 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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 border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <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>Free </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&uuml;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></td>
 <td></td>
 <td></td>
 <td>Table 1. Required reagents and instruments.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Live DNA Stain Solution (10 ml):
 1 ml 10X Ca2+ Mg2+ free PBS (pH7.2) 
 9 ml 10X Trypsin-EDTA (Sigma) 
 5 &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 Drosophila cells.)
 10X Ca2+ Mg2+ free PBS (pH7.2): 1.37M NaCl, 27 mM KCl, 100mM Na2HPO4 (dibasic), 20mM KH2PO4 (monobasic) adjusted to pH 7.2
 </td>
</tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td><table border="1">
 <tbody>
 <tr>
 <td></td>
 <td>FSC</td>
 <td>SSC</td>
 <td>BL1</td>
 <td>VL1</td>
 </tr>
 <tr>
 <td>Threshold (x1000)</td>
 <td>100</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 </tr>
 <tr>
 <td>Voltage (mV)</td>
 <td>2950</td>
 <td>4250</td>
 <td>1800</td>
 <td>1150</td>
 </tr>
 </tbody>
 </table>
Table 2. Threshold and voltage setting for the analysis in Figure 2.</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 ...

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

13.5K 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

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

باستخدام مايكرو سيستمز الكهربائية والميكانيكية (ممس) لتطوير أدوات التشخيص

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

لإعداد ذبابة الفاكهة الأجنة لايف التصوير باستخدام بروتوكول إسقاط الشنق

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

يعيش تصوير ذبابة الفاكهة السوداء البطن هجرات كرية دموية الجنينية

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

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 ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

تقييم التعبير GFP واستمرار استخدام يستند إلى صورة طالي عداد الكريات

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

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.

يعيش التصوير من تخليص الخلية أفكارك أثناء<em&gt; ذبابة الفاكهة</em&gt; مرحلة التطور الجنيني

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

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

والطماطم / GFP-FLP / FRT الطريقة للتصوير لايف من الفسيفساء الكبار<em&gt; ذبابة الفاكهة</em&gt; خلايا مستقبلة للضوء

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

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 ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

انخفاض الجاذبية المظاهرات الأجهزة البيئة في نموذج المنمنمة تدفق عداد الكريات ورفيق ميكروفلويديك تقنية خلط

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

مقايسة الامتصاص الكولسترول استخدام الخلايا الحية التصوير التحليل مع خلية المراقبة الصحية

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...