الكتاب نود أن نشكر المعاهد الكندية لأبحاث الصحة (CIHR) وجمعية السرطان الكندية للحصول على تمويل أبحاث الخلايا دورة في المختبرات الخاصة بهم. MJC هي المستفيدة من منحة MD / دكتوراه من CIHR. MJC والماجستير هي أعضاء في برنامج التدريب CaRTT.

<ol>
	<li>Dolbeare, F., Gratzner, H., Pallavicini, M. G., Gray, J. W. <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+measurement+of+total+DNA+content+and+incorporated+bromodeoxyuridine.">Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine.</a> Proc. Natl. Acad. Sci. U. S. A. 80, 5573-5573 (1983).</li><li>Givan, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+an+introduction.">Flow cytometry: an introduction.</a> Methods. Mol. Biol. 699, 1-1 (2011).</li><li>Kalejta, R. F., Brideau, A. D., Banfield, B. W., Beavis, A. 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في تجربتنا ، والنجاح مع هذه التقنيات تعتمد على مفتاح التحكم قليلة والظروف التجريبية. واحد هو إقامة سيطرة المتكاثرة بشكل غير متزامن لاستخدامها في هذه التجارب. هذه السيطرة يخدم ثلاثة أغراض هامة. الأولى ، فإنه يضمن ظروف ثقافة المستخدمة لجميع العينات التجريبية هي كافية لدعم الانتشار المتواصل في غياب العلاج. هذه العينة أيضا يخدم الغرض من مراقبة إيجابية لتلطيخ منهجية لضمان أن يتم الكشف عن خلايا CD20 BrdU أو إيجابية عندما كانت موجودة. أخيرا ، يتم استخدام هذه العينة لمعايرة عداد الكريات التدفق. ويمكن استخدام هذا النموذج التحكم لضبط شدة propidium تلطيخ يوديد للكشف عن خلايا 2N و4N في 200 و 400 على التوالي. وعلاوة على ذلك ، يمكن ضبط حساسية الكشف عن BrdU أو CD20 بحيث تتركز إشارات سلبية وإيجابية كما هو مبين في الأرقام و1A 2A. عندما يكون جميع العينات تركزا مماثلا من الخلايا في PI - RNase الحل ، ثم هناك حاجة إلى بعض التعديلات في عداد الكريات كما يتم تشغيل عينات لاحقة. هذا مهم لأسباب المبينة في الشكل. 4B 5B وقوية حيث تراكم يخلق أساسا G1 G1 ذروة واحدة يمكن ان يساء تفسيرها على أنها G2 / م. التجارب أيضا جعل ممثل نقطة هامة أخرى. بادئ ذي بدء ، فإنها تثبت أن BrdU امتصاص ووضع العلامات يمكن أن تتفاوت بين أنواع الخلايا. لهذا السبب من المهم تجريبيا لتحديد طول النبض والشروط اللازمة للكشف عن تلطيخ الخلايا على نحو كاف في S - المرحلة. عموما ، يمكن أن توصف أنواع الخلايا التي مزدوجة في الثقافة في 24 ساعة أو أقل مع BrdU في ساعة واحدة. قد أبطأ أنواع الخلايا المتزايدة تتطلب تعد البقول وتعديلات على الشروط تلوين الأجسام المضادة ومدتها. خلايا MCF10A هي مثال ممتاز في هذا الصدد كما وصفت لهم BrdU لمدة أربع ساعات والملون مع التركيز على ضعف مستوى الأجسام المضادة لمدة أربعة أضعاف طويلة. محققونيجب أن نكون حذرين بحيث لا يتجاوز 6 ساعات من وضع العلامات BrdU حتى مع تزايد الخلايا ببطء شديد لأن ذلك يمكن أن يؤدي خطأ إلى إدراج G2 / M الخلايا في السكان S - المرحلة. ثانيا ، في المقارنة بين آثار p27KIP1 التعبير مع تلك TGF - β1 ، فمن الواضح أن P27 يمكن أن تحدث في أي تراكم أو المراحل G1 G2 / M - β1 TGF بينما يشير يؤدي الى اعتقال G1. الوسائل التقليدية للكشف عن انتشار مثل إدماج H - 3 ثيميدين والتلألؤ عد غير قادر على التمييز بين هذه الاحتمالات. في بروتوكول لدينا بديل نظهر للكشف عن مجموعة من السكان فرعية من الخلايا في عدد السكان أكبر untransfected. من المهم تحديد تجريبيا الصحفي الأكثر ملاءمة للكشف عن خلايا transfected. تجربتنا في بعض أنواع الخلايا إما عن سطح الخلية علامات سيئة ، أو لا يمكن المرور عليها بشكل صحيح إلى غشاء البلازما ، مما أدى إلى عدم القدرة على الكشف عن خلايا transfected. Likewise ، وليس كل الخلايا تحمل شكل غشاء ملزمة للGFP. وينبغي أن يتم الاختيار لمراسل أنسب من خلال تقييم كفاءة ترنسفكأيشن لمراسل وكذلك الكثافة النسبية للتعبير مقارنة مع الضوابط untransfected. من الناحية المثالية ، سيتم تعبير مغايرة لهذه الجزيئات جيد التحمل ، وهذا يوحي بأن ليس لديهم علامات على أي تأثير على توزيع دورة الخلية. القيد واحد من هذه الأنواع من نهج التدفق الخلوي هو أنها الوحيدة القادرة على وضع فرة نسبية من مراحل دورة الخلية مقارنة مع بعضها البعض. لهذا السبب فهو تعبير عن حالة غامضة P27 في الشكل 3 يدفع حقا الاعتقال في G1 و G2 / M ، أو إذا كان مجرد يبطئ تقدم من خلال هذه المراحل بالنسبة إلى S - المرحلة. ويمكن تحقيق المزيد من هذه الاحتمالات من خلال معالجة عينة من الخلايا المتوازية مع مثبط الإنقسامية مثل nocodazole ، أو G1 / S مثبط مثل aphidicolin. منذ إنشاء هذه الأدوية دوميناNT الاعتقال في المرحلة M - S - أو أوائل مرحلة على التوالي ، وسوف تتراكم ببطء الخلايا المتكاثرة في اعتقال نقطة المخدرات التي يسببها. لسوف الخلايا سبيل المثال ألقي القبض عليه في G1 بسبب التعبير PRB البقاء في G1 على الرغم من العلاج في حين أن الخلايا nocodazole السيطرة سوف تتراكم في المرحلة M - 13. أخذت معا ، وهذه المناهج التجريبية تقترح منهجية مرنة يمكن تطبيقها على مجموعة واسعة من الأسئلة أبحاث الخلايا الثديية دورة. فإنها يمكن بسهولة اكتشاف تغيرات في تطور دورة الخلية وتحديد الاختلافات عند مقارنة مع الضوابط.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف / المعدات </th><th> شركة </th><th> فهرس العدد </th><th> تعليق (اختياري) </th></tr><tr><td> كاشف خلية الانتشار وصفها </td><td> GE أمرشام </td><td> RPN201 </td><td></td></tr><tr><td> أجسام مضادة للBrdU </td><td> العلوم البيولوجية دينار بحريني </td><td> 347580 </td><td></td></tr><tr><td> أجسام مضادة للماوس FITC مترافق </td><td> ناقلات مختبرات </td><td> FI - 2000 </td><td></td></tr><tr><td> مرشحات سلالة الخلية </td><td> فالكون دينار بحريني </td><td> 352235 </td><td></td></tr><tr><td> أجسام مضادة للCD20 </td><td> العلوم البيولوجية دينار بحريني </td><td> 347673 </td><td></td></tr><tr><td> دورة برامج متعددة </td><td> نظم تدفق الفينيق </td><td> N / A </td><td></td></tr></tbody></table>

analysis of cell cycle position in mammalian cells

تنظيم تكاثر الخلايا والأنسجة المركزية إلى التشكل خلال تطوير الكائنات المتعددة الخلايا. وعلاوة على ذلك ، وفقدان السيطرة على انتشار الخلايا تكمن وراء أمراض من أمراض مثل السرطان. على هذا النحو هناك حاجة كبيرة لتكون قادرة على التحقيق في تكاثر الخلايا وquantitate نسبة الخلايا في كل مرحلة من مراحل دورة الخلية. كما أنها ذات أهمية حيوية لتحديد الخلايا التي يتم indistinguishably تكرار الحمض النووي في غضون عدد أكبر من السكان. منذ قرار يرصد خلية لتتكاثر في المرحلة G1 مباشرة قبل الشروع في تركيب الحمض النووي والتقدم خلال الفترة المتبقية من دورة الخلية ، واكتشاف تركيب الدنا في هذه المرحلة يسمح لعزم لا لبس فيه للوضع القائم للتنظيم نمو الخلايا في الثقافة التجارب. يمكن أن يكون محتوى الحمض النووي في الخلايا quantitated بسهولة التدفق الخلوي للخلايا ملطخة يوديد propidium ، صبغة الفلورسنت الإقحام الحمض النووي.وبالمثل ، يمكن quantitated نشطة من قبل خلايا الحمض النووي التوليف زراعة في حضور ثيميدين المشعة ، حصاد الخلايا ، وقياس النشاط الاشعاعي في إدراج جزء غير قابل للذوبان حمض an. لدينا خبرة كبيرة مع تحليل دورة الخلية وتوصي باتباع نهج مختلف. نحن بالتحقيق في انتشار الخلايا باستخدام bromodeoxyuridine / fluorodeoxyuridine (مختصر كمجرد BrdU) تلطيخ يكشف عن إدراج هذه النظير الثايمين في الحمض النووي توليفها في الآونة الأخيرة. التوسيم وتلطيخ الخلايا مع BrdU ، جنبا إلى جنب مع تلطيخ DNA مجموع يوديد propidium وتحليلها من جانب التدفق الخلوي 1 يوفر المقياس الأكثر دقة من الخلايا في مراحل مختلفة من دورة الخلية. هو الأسلوب المفضل لدينا لأنه يجمع بين اكتشاف تركيب الدنا نشطة ، من خلال تلطيخ القائمة على الضد BrdU ، مع محتوى الحمض النووي من مجموع يوديد propidium. وهذا يسمح لفصل واضح للخلايا في مرحلة مبكرة من G1 S ، أو S في وقت متأخر من مرحلة G2 / م.وعلاوة على ذلك ، يمكن استخدام هذا النهج لتحقيق العديد من الآثار منبهات مختلفة من الخلايا وكلاء الصيدلانية على التقدم من خلال تنظيم هذه المراحل دورة الخلية المختلفة. في هذا التقرير وصفنا طرق لوضع العلامات وتلطيخ الخلايا المستزرعة ، وكذلك من خلال تحليل التدفق الخلوي. نحن تشمل أيضا أمثلة تجريبية لكيف يمكن استخدام هذه الطريقة لقياس الآثار المترتبة على تثبيط نمو إشارات من السيتوكينات مثل TGF - β1 ، ومثبطات التكاثري مثل مثبط كيناز cyclin التابعة ، p27KIP1. علينا أيضا أن تشمل بروتوكول بديل يسمح لتحليل الموقف في دورة الخلية من الفئات السكانية الفرعية داخل الخلايا أكبر ثقافة 5. في هذه الحالة ، ونحن لشرح كيفية الكشف عن اعتقال خلية في دورة transfected خلايا الشبكية مع الجينات حتى عندما فاق عدد كبير من الخلايا untransfected في الثقافة نفسها. هذه الأمثلة توضح العديد من الطرق التي الحمض النووي وتلطيخويمكن استخدام التدفق الخلوي وتكييفها للتحقيق في المسائل الأساسية للسيطرة دورة الخلية الثديية.

Watch this Scientific Journal Video about Analysis of Cell Cycle Position in Mammalian Cells at JoVE.com

Analysis of Cell Cycle Position in Mammalian Cells

تحديد موقف دورة الخلية من مجموعة من الخلايا ، أو فهم الكيفية التي تؤثر على انتشار الاشارات ، يمكن قياس التدفق الخلوي بسهولة باستخدام هذا البروتوكول. نحن التقرير مقاربة بسيطة التجريبية لخلايا التلوين وقياس موقفهم في دورة الخلية.

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 ...

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59.9K Views.  University of Western Ontario. تحديد موقف دورة الخلية من مجموعة من الخلايا ، أو فهم الكيفية التي تؤثر على انتشار الاشارات ، يمكن قياس التدفق الخلوي بسهولة باستخدام هذا البروتوكول. نحن التقرير مقاربة بسيطة التجريبية لخلايا التلوين وقياس موقفهم في دورة الخلية.

Video: Analysis of Cell Cycle Position in Mammalian Cells

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

التصوير خلايا الثدييات على المدى الطويل باستخدام الميكروسكوب واسعة الميدانية

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

نبض مطاردة تحليل سلاسل السكر N - الموصولة من جليكوبروتينات في خلايا الثدييات

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

تحليل مزدوج الجديلة إصلاح الحمض النووي (DSB) فاصل في خلايا الثدييات

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

esiRNA بعثة لفحص رني في خلايا الثدييات

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

تحليل حركية مرنا الصادرات النووية في خلايا الثدييات التي Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

تحليل التعبير عن الأنواع الفرعية رابط-هيستون الثدييات

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

تصور mRNAs والشبكة الإندوبلازمية في خلايا الثدييات المترجمة

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

قياس مستويات هيم في تخليق خلايا الثدييات

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

التصوير المحلية الكالسيوم<sup&gt; 2+</sup&gt; الإشارات في خلايا الثدييات مثقف

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...