This work was supported by National Institutes of Health grants GM 100201 to I.F.S, and GM 048071 and GM 065830 to I.P.

Biz insan nöroblastom SH-SY5Y hücrelerinde yerel Ca 2 + olayları görüntüleme ayrıntılı prosedürler sunuyoruz. Bu prosedürler, çoğu hücre tipinde 8-10 görüntü hücre içi Ca + 2 sinyallere uyarlanabilir. Hücreler 1. Hazırlık <ol><li> Kültür hücreleri tedarikçinin Web sitesinde listelenen talimatlara göre yansıması için. </li><li> Görüntü vermeden önce birkaç gün, hasat hücreleri, 1 ml 0.25% tripsin / EDTA (2-3 dakika) kullanılarak bir doku kültürü şişesi içinde büyütülür. Dekolmanı ardından, tripsin inaktive kültür ortamı eşit hacimde ekleyin. </li><li> Tabağı başına 3-7 x 10 4 hücre yoğunluğunda cam alt görüntüleme kapları içine bir hemasitometre ve tohum hücreleri kullanılarak bir hücre sayımı yapın. Görüntüleme deneyleri için hücreleri seçin onlar% 60 izdiham (2-3 gün) ulaştığınızda. </li></ol> Çözümler ve Reaktifler 2. Hazırlık <ol><li> Tamponlu Ca 2+ çözülebilmekle HEPES hazırlayın(mM) oluşan tuz çözeltisi (Ca 2 + -HBSS): 135 NaCl, CaCI2, 1 MgCl2, 10 HEPES, 10 glukoz 5.4 KCI, 2; pH = 7.4 NaOH ile oda sıcaklığında (RT). 4 ° C &#39;de glikoz ve mağaza eklenmeden Ca 2 + -HBSS hazırlanması; kullanımdan hemen önce solüsyona glikoz ekleyin. </li><li> Floresan Ca2 + göstergesi Cal-520 (1 mM) zara nüfuz edici formları çözünür, Cı-IP 3 (200 uM) ve EGTA% 20 Pluronic F-127 içeren bir dimetil sülfoksit (100 mM), (DMSO). -20 ° C de, nem ve ışıktan korunan Eppendorf tüpleri ve mağaza içine, bu reaktif maddeleri bir kısım. </li></ol> 3. Yükleme Hücreler Membran-geçirgen, Cal-520 ci-IP 3 ve EGTA <ol><li> Ca + 2 -HBSS sırasıyla 5 uM ve 1 uM nihai bir konsantrasyona kadar zara nüfuz edici Cal-520 ve CI-IP 3 stok çözelti seyreltilerek &quot;yükleme tamponu&quot; hazırlayın. </li><li> Aspire culture medya ve Ca 2+ -HBSS üç kez medya değiştirerek hücreleri durulayın. </li><li> Ca2 + çıkarın -HBSS ve karanlıkta, oda sıcaklığında 60 dakika süre ile yükleme tampon maddesi içinde hücrelerin inkübe edin. </li><li> Yükleme tamponu çıkarın ve Ca 2+ -HBSS üç kez medya değiştirerek hücreleri durulayın. <ol><li> Arzu edildiği takdirde, bu aşamada, daha sonra yük Ca 2 + -HBSS 5 mM&#39;lik bir son konsantrasyona kadar stok çözelti seyreltilerek EGTA / AM ile hücreler ve karanlıkta, oda sıcaklığında, 30-60 dakika boyunca kuluçkalayın. Bu inkübasyonun ardından, 3.5 adıma devam etmeden önce, hücreler Ca + 2 -HBSS ile üç kez yıkayın. </li></ol></li><li> Yüklenen reaktifler de-esterifikasyonu için izin vermek için 30 dakika süre ile Ca + 2 -HBSS hücreleri inkübe edin. </li><li> Görüntüleme hemen önce, son inkübasyon sırasında banyoya sızan olabilecek boya kaldırmak için &quot;taze&quot; Ca 2 + -HBSS Ca 2+ -HBSS değiştirin. </li></ol> 4. Ca 2+ </sup&gt; Image Acquisition <ol><li> 100X APO TIRF objektif üzerine daldırma yağ küçük bir damla (NA 1.49) yerleştirin. </li><li> Ters mikroskop sahnede görüntüleme çanak monte ve iletilen ışık kullanarak odak noktası haline getirmek hücreleri. Bu kayıt sırasında hareket etmesini önlemek için görüntüleme çanak sabitlemek için önemlidir. </li><li> Yüksek hızlı EMCCD kamera kullanılarak yayılan floresan (λ&gt; 510 nm) Cal-520 heyecanlandırmak ve toplamak için bir 488 nm lazer ile hücreleri aydınlatın. NOT: mikroskop faaliyet yazılım paketi kullanıcı lazer ışını ya (&quot;WF&quot; uyarma için) daha merkezi ya da (TIRF uyarma için) objektif arka diyafram aşırı kenarında tanıtılacak sağlayan bir odaklama lens çevirmek için izin verir. </li><li> EMCCD yazılımı kullanarak, yerel Ca 2 + olayları yakalamak için gerekli zamansal çözünürlükte veri toplamak için tam 512 x 512 piksel görüntüleme alanını azaltır. NOT: Örneğin, orta dörtlü256 x 256 piksel edinimi 66 fps&#39;ye satın alma oranını hızlandırır. Bundan başka, bir izole edilmiş Bitki Mod fonksiyonu kullanılarak 500 fps EMCCD kameranın kare hızını arttırmak için de mümkündür. </li><li> Otomatik, bazal aktivite birkaç saniye kayıt hücrelere UV flaş teslim ve başka 10-30 saniye kayıt devam etmek yazılım yapılandırın. NOT: UV flaş mikroskop arka limanında dikroik ayna yansıtan bir UV ile tanıtıldı Xenon ark ışık kaynağı kullanılarak teslim edilir. UV flaş süresi ve yoğunluğu yerel Ca 2 + olayların İstenen frekansı uyandırmak için ampirik ayarlanır. </li><li> Görüntü off-line analiz için yığılırken dosyaları kaydedin. </li></ol> 5. Otomatik Ca 2+ Görüntü Analizi NOT: Bu görüntülemek, süreç ve birçok ticari yazılım paketleri kullanılarak yakalanan verileri analiz etmek mümkündür. Ancak, hızla tanımlanması bir otomatikleştirmek için bir algoritma geliştirdikYerel Ca 2 + sinyalleri d analizi. Bu algoritma, mekansal ve zamansal filtre ayrıntılı bir tarifi, mesafe kadar taşınmış / F 0 ve kimlik / analiz rutinleri üretimi 11 bulunabilir. Bu algoritma, açık kaynak kodlu bir yazılım platformu Python üzerinde çalıştırmak için geliştirilmiştir, tarafından, birlikte örnek deneysel verilere ve detaylı kullanıcı talimatları ile elde edilebilir, e-posta gelen yazar (ismith@uci.edu). <ol><li> Dosyalarını dönüştürme çoklu uçağın .tiff dosya formatına özel yazılmış algoritma ithal edilecek. </li><li> Analiz algoritması içeren klasörü bulun ve çift tıklayın RUN.EXE. </li><li> .tiff Dosyayı seçin analiz edilecek. </li><li> Açık iletişim kutusunda analiz parametrelerini seçin. Örnek verileri için varsayılan parametreleri Şekil 2A bakın. </li><li> Siyah seviyesini belirlemek ya n yapar görüş alanının bir kısmına imleci hareket ettirerek görüntü yığını ofset edilecekot hücreyi içerirler (Şekil 2B bakınız) veya elle &#39;Set Siyah Seviye&#39; istemini kullanarak siyah seviyesi girerek. </li><li> Yazılım (Şekil 2C-F) tarafından analiz aşağıdaki ekranda dört pencere gözlemleyin. C hücrelerinden floresan istirahat bir tek renkli görüntü analiz ediliyor. Bu resmin üzerine bindirilmiş Beyaz kareler olaylara takılı iki boyutlu Gauss fonksiyonları sentroid pozisyonları olarak belirlenen etkinlik konumları vardır. </li><li> Her olay yere tıklayın ya da olaylar arasında dolaşmak için imleç tuşlarına basın. Bunu yaparken üzerine arka plan çıkarılır, Gauss penceresi D. bu siteler güncelleme her floresan oranı değişiklikleri (mesafe kadar taşınmış / F 0) düzeltti NOT: Kırmızı olayları komşu sitelere lokalize faaliyetten &#39;sızdırma-through&#39; o belirli sitesinden kaynaklanan ve kararlı olduklarını vurguladı. Seçilen yerinde floresan sinyal algılama aştı nerede alt mavi iz gösterirSiyah çizgi söz konusu sitesinden kaynaklanan olayları tanımlar ise eşik, (kırmızı karşılık gelen olayları vurgulanan). </li><li> Bir kırmızı tıklayın olayın zamansal evrimi görüntüler pencere E, ve birlikte takılan Gauss fonksiyonu mekansal profili ile, olayın mekansal profil zamanı boyunca ortalama görüntüler pencere F güncellemek için olay vurgulanır. </li><li> El eserler analiz reddedilmesi, böylece tespit edilmiştir olaylarını. Sağ kırmızı tıklayarak tür olayları silmek olayları vurgulanır. NOT: Bizim ellerde, Ca tek bir IP 3 R kanallardan 2+ akı kadar taşınmış / F 0 yaklaşık 0.11 ile sinyalleri çağrıştırıyor, bu nedenle bu ölçüde daha küçük herhangi bir tespit &#39;etkinlikleri&#39; yanlış pozitif olması muhtemeldir. </li><li> Kaydet CellR ilgi hücrenin etrafında çizim ve &#39;seçerek hücre bazında bir veri veya ihracat&#39; Excel kaydet &#39;seçerek İhracat verileri17 ;. NOT: Veriler, orijinal görüntü yığınıyla aynı klasöre kaydedilir. </li></ol>

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The authors declare that they have no competing financial interests.

Biz burada açıklamak floresan Ca 2 + göstergeleri kullanarak kültürlü memeli hücrelerinde yerel Ca 2 + olayları görüntüleme için protokolleri. Ayrıca, kimlik ve elde edilen verilerin analizi otomatik bir sezgisel kullanıcı arayüzü ile bir algoritma tanımlamaktadır. Burada tarif edilen prosedür floresan Ca 2 + göstergesi Cal-520 kullanmak, ancak Flu-3, Flu-4, Flu-8 ve Oregon Yeşil BAPTA-1 gibi diğer birçok Ca 2 + duyarlı boyalar görüntü Ca yeterince ...

Kalsiyum iyonları (Ca 2+) yayg gen ifadesi, sekresyon ve sinaptik plastisite 1 uzun ömürlü değişiklikler dahil olmak üzere biyolojik süreçlerin, çeşitli bir yelpazede düzenler. Ca 2 + Böyle farklı bir şekilde hareket hangi aracılığıyla bir yolu Ca 2+ farklı mekansal ve zamansal desenleri ile bir hücre oluşturabilir sinyalleri. Sitozolik örnek küresel yükselme düz kas dokusu 2 ise daha küçük [Ca 2+] tetik daralma, lokalize geçici yükselmeler (yerel Ca 2 + mikro bölgeler) için öğrenme ve bellek 3 için gerekli gen ifadesini teşvik. Ücretsiz sitozolik [Ca 2 +] ~ 100 nM dinlenme tutulur, fakat hızla Ca ile sitozolüne Ca 2+ akını aşağıdaki birkaç mikro-molar plazma zarı ve yanında bulunan 2 + -geçirgen iyon kanallarını çıkabilir hücre içi s Ca 2+ kurtuluştores. Bizim laboratuvar endoplazmik retikulum (ER) zarında bulunan Ca 2 + bırakma kanalı oluşturan inositol 1,4,5-trifosfat alıcısı (IP 3 R), odaklanır. Reseptör siteleri aktive sitozolik hem IP 3 ve Ca 2+ bağlanması üzerine, IP 3 R kanalı Ca 2 + ER lümeninde tecrit kurtarmak açılır. Ca 2+ salınımı uzamsal (Ca 2 + puf 4) 2+ Ca yerel sitozolik mikro oluşturmak için IP 3 Rs küçük bir küme sınırlı kalabilir veya IP 3 Rs komşu kümelerinin yakınlığı bağlı olabilir Ca 2+ indüklediği Ca 2 + -salınımı (YÜÇAM) 5,6 süreciyle birden puf siteleri işe bir hücre boyunca yaymak. Gelişmiş mikroskobu IMAGI ile birleştiğinde Tsien 7 Roger tarafından geliştirilen floresan küçük moleküllü Ca 2+ gösterge boyaların tanıtımı,teknikleri ng, Ca 2+ sinyalizasyon anlayışımızı büyük katkı sağlamaktadır. Mikroskopi için kullanılan kameralar son gelişmeler şimdi böyle görülmemiş mekansal ve zamansal çözünürlüğe sahip ponponları gibi geçici yerel Ca 2 + olayları görüntüleme için izin verir. Şu anda mevcut EMCCD kameralar&gt; 500 kare saniye -1 (fps) ve tamamlayıcı metal oksit yarı iletken yeni nesil 128 x 128 piksel ile görüntüleme sağlayan (CMOS) kameralar biraz daha yüksek pahasına yüksek piksel çözünürlük, ve hatta daha hızlı sağlamak gürültü seviyeleri. Toplam iç yansıma (TIRF) mikroskopi ile birlikte bu görüntüye artık tek Ca 2 + kanal olayları 8,9 mümkündür. Impracticable manuel işleme, görsel kimlik ve analiz hale ve otomatik algoritmalarının geliştirilmesi yapma yükümlülüğü büyük veri setleri (min başına yaklaşık 1Gb) oluştururken Bu yaklaşım, aynı zamanda kanal / olayların yüzlerce görüntüleme için izin verir. 2 + göstergeleri kullanarak sağlam memeli hücrelerinde yerel Ca 2 + sinyalleri görüntüleme prosedürlerini ve protokolleri sunuyoruz. Biz daha TIRF ve geleneksel geniş alan epi-floresan (WF) mikroskopi hem görüntülü yerel Ca 2 + olayların belirlenmesi ve analizi otomatik açık kaynak kodlu ortam Python geliştirilen bir algoritma göstermek. Biz IP Ca 2 + sinyalleri -generated 3 bağlamında bu yaklaşımları açıklamak rağmen, [Ca 2 +] Ca çeşitli ya yüzeye bulunan 2+ -geçirgen iyon kanallarını yayılan sitozolik yerel değişiklikleri incelemek için kolayca mükellef olan Membran ya da hücre içi organellerin 8-10.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:813px;">Name</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalogue No.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Neuroblastoma SH-SY5Y cells</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cal-520/AM</td>
			<td>AAT Bioquest Inc.</td>
			<td>21130</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ci-IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4.5-dimethoxy)benzyl-myo-Inositol 145-trisphosphate-Hexakis(propionoxymethyl)</td>
			<td>SiChem</td>
			<td>cag-iso-2-145-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO/20% pluronic F127</td>
			<td>Invitrogen</td>
			<td>P-3000MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA/AM</td>
			<td>Invitrogen</td>
			<td>E-1219</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom imaging dishes</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</td>
		</tr>
	</tbody>
</table>

imaging local ca2+ signals in cultured mammalian cells

Sitosolik Ca + 2 iyonları, gen transkripsiyonu, elektriksel uyanlabilirliği ve hücre çoğalması gibi çok kapsamlı işlemlerin kontrol, hemen hemen tüm hücre tiplerinde hücresel aktivitesi sayısız yönlerini düzenleyen. Ca 2 + sinyalizasyon çeşitliliği ve özgünlüğü yerel geçici Ca 2 + microdomains dakika süre boyunca meydana gelen hücre çapında titreşimler ve dalgalar arasında değişen Ca 2 + sinyalleri farklı zaman ve mekansal ölçekler üzerinde hareket oluşturulur hangi mekanizmalar, türemiştir (Ca 2+ ponponları) kalıcı milisaniye. Elektron son gelişmeler CCD (EMCCD) kameralar artık&gt; 500 kare saniye -1 (fps) oranlarında 128 x 128 piksel mekansal çözünürlüğe sahip yerel Ca 2 + sinyalleri görüntüleme için izin çarpılır. Bu yaklaşım son derece paralel ve tek bir deneyde kanallar veya puf sitelerin yüzlerce eşzamanlı izlenmesini sağlar. Ancak, verilerin büyük miktarlarda üretilen (yaklaşık 1 Gb per dk) 2+ olayları uygulanamaz yerel Ca görsel kimlik ve analiz kılıyor. Burada tarif ve satın alma, algılama için prosedürleri göstermek ve geniş alan epi-floresan (WF) ve toplam iç yansıma ikisini de kullanarak Ca 2 + göstergeleri ile yüklenen sağlam memeli hücrelerinde Ca 2+ sinyalleri aracılı 3 yerel IP analizi floresan (TIRF) mikroskopi. Ayrıca, bu yerel Ca 2 + sinyallerin belirlenmesi ve analizi otomatik açık kaynak yazılım ortamı Python içinde geliştirilen bir algoritma tarif. algoritma alt piksel çözünürlüğe sahip Ca 2+ sürümü siteleri lokalize; veri kullanıcı gözden sağlar; ve bir Excel uyumlu tabloda genlik ve kinetik verileri ile birlikte floresan oranı sinyallerinin zaman dizilerini çıktılar.

Şekil 1A, CI-IP 3 yüklü insan nöroblastoma SH-SY5Y hücrelerinin Cal-520 floresan dinlenme bir WF görüntüsünü gösterir. Fotoğraf-sürüme 100 milisaniye UV flaş bu hücrelerin Pozlama i-IP 3 (Şekil 1A beyaz çevreler tarafından not) ayrık siteleri geçici Ca 2 + ponponları ortaya çıkardı. Bu sitelerde ölçülen Floresan izleri yavaş yavaş bırakma sitesinden yayılan 2+ Ca gibi bir çok yavaş düşen faz (Şekil ...

Watch this Scientific Journal Video about Imaging Local Ca2+ Signals in Cultured Mammalian Cells at JoVE.com

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.

Görüntüleme Yerel Ca<sup&gt; 2+</sup&gt; Kültürlenmiş memeli hücrelerinde sinyaller

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

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JoVE Journal

Biology

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

10.7K Görüntüleme.  University of California, Irvine. 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.

Video: Imaging Local Ca2+ Signals in Cultured 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.

Geniş Alan Mikroskop kullanarak Uzun vadeli Görüntüleme Memeli Hücreleri

Biyoloji

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Görüntüleme Ca Teknikleri 2 + İnsan Sperm sinyal

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

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.

Memeli Hücreleri RNAi Tarama MİSYON esiRNA

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Canlı hücreler Mağaza çalışan Kalsiyum Giriş Floresans tabanlı Ölçümü: Kültür Kanseri Hücre gelen İskelet Kası Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

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

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Kültür Hücrelerinin retikulum Subdomain&#39;ler Görselleştirme

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Dinamik Ca Otomatik Analizi<sup&gt; 2 +</supGörüntü Dizilerinde&gt; Sinyaller

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Kültür Memeli Hücrelerde Biyolojik tanımında ubikitin ve Ubiquitin benzeri Sistemi Regülatörleri tanımak siRNA Eleme

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

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 ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.