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.

Nós apresentamos os procedimentos detalhados de imagem Ca 2+ eventos locais em neuroblastoma humano células SH-SY5Y. Estes procedimentos podem ser adaptados para a imagem de Ca 2+ intracelular sinais em muitos tipos de células 8-10. 1. Preparação de células <ol><li> Cultura as células a ser trabalhada de acordo com as instruções constantes no site do seu fornecedor. </li><li> Alguns dias antes da imagiologia, colheita células cultivadas num frasco de cultura de tecidos com 1 ml de 0,25% de tripsina / EDTA (2-3 min). Após separação, adicionar um volume igual de meio de cultura para inactivar a tripsina. </li><li> Executar uma contagem de células utilizando um hemocitómetro e as células em pratos de semente de imagem de fundo de vidro, a uma densidade de 3-7 x 10 4 culas por prato. Selecione as células para experimentos com imagens quando chegar a 60% de confluência (2-3 dias). </li></ol> 2. Preparação de Soluções e Reagentes <ol><li> Prepare uma CA 2+ molecular contendo tampão HEPESsolução de sal de (Ca 2+ -HBSS) composto por (mM): 135 NaCl, 5,4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, e 10 de glucose; pH = 7,4 à temperatura ambiente (TA) com NaOH. Prepare Ca 2+ -HBSS sem a adição de glicose e armazenar a 4 ° C; adicionar a solução de glicose imediatamente antes da utilização. </li><li> Solubiliza-se formas de membrana permeante do Ca 2+ indicador fluorescente Cal-520 (1 mM), ci-IP 3 (200 uM), e EGTA (100 mM) em sulfóxido de dimetilo (DMSO) contendo 20% de Pluronic F-127. Alíquota estes reagentes em tubos Eppendorf e armazenamento, protegido da humidade e da luz, à temperatura de -20 ° C. </li></ol> 3. Carregando Cells com uma membrana de permeant Cal-520, ci-IP 3 e EGTA <ol><li> Preparar o &quot;tampão de carga&quot; diluindo soluções de reserva de permeante da membrana Cal-520 e ci-IP 3 até uma concentração final de 5 uM e 1 uM, respectivamente, em Ca 2+ -HBSS. </li><li> Aspirar cumedia LTURE e lavar as células através da substituição de mídia com Ca 2+ -HBSS três vezes. </li><li> Remover Ca 2+ -HBSS e incubar as células em tampão de carga, durante 60 minutos à temperatura ambiente no escuro. </li><li> Remover tampão de carregamento e enxaguar células, substituindo mídia com Ca 2+ -HBSS três vezes. <ol><li> Se desejado, nesta etapa, as células de carga com EGTA / AM por diluição da solução de reserva até uma concentração final de 5 uM em Ca 2+ -HBSS e, em seguida, as células incubar durante 30-60 minutos à temperatura ambiente no escuro. Após esta incubação, lavar as células três vezes com Ca 2+ -HBSS antes de prosseguir para o passo 3.5. </li></ol></li><li> Incubar as células em Ca 2+ -HBSS durante 30 min para permitir a desesterificação de reagentes carregados. </li><li> Imediatamente antes de imagem, substitua Ca 2+ -HBSS com &quot;fresco&quot; Ca 2+ -HBSS, a fim de remover qualquer corante que pode ter vazado para o banho durante a incubação final. </li></ol> 4. Ca 2+ </sup&gt; Aquisição de Imagem <ol><li> Coloque uma pequena gota de óleo de imersão para o objetivo 100X APO TIRF (NA 1,49). </li><li> Montar o prato de imagem na fase do microscópio invertido e trazer as células em foco utilizando luz transmitida. É importante proteger o prato de imagem para impedir o movimento durante a gravação. </li><li> Iluminar as células com um laser de 488 nm, a fim de excitar Cal-520 e recolher a fluorescência emitida (λ&gt; 510 nm) usando uma câmara EMCCD alta velocidade. NOTA: O pacote de software que opera o microscópio permite ao utilizador para traduzir uma lente de focagem, permitindo que o feixe de laser a ser introduzido quer no bordo extremo da abertura traseira objectivo (por excitação TIRF) ou mais centralmente (para a excitação &quot;WF&quot;). </li><li> Usando o software EMCCD, reduzir o campo de imagem a partir da plena 512 x 512 pixels, a fim de coletar dados na resolução temporal necessário para capturar Ca 2+ eventos locais. NOTA: Por exemplo, quad centeraquisição de 256 x 256 pixels acelera a taxa de aquisição de 66 fps. Também é possível aumentar ainda mais a taxa de quadros da câmera EMCCD para 500 fps usando uma função Modo de recorte isolado. </li><li> Configure o software para gravar automaticamente alguns segundos de atividade basal, entregar um flash UV para as células e continuar a gravação para outra 10-30 seg. NOTA: Um lampejo de UV é entregue utilizando uma fonte de luz de arco de xénon introduzido através de um espelho dicróico UV reflectindo na porta traseira do microscópio. A duração e intensidade do lampejo de UV é ajustado empiricamente para evocar uma frequência desejada de Ca 2+ acontecimentos locais. </li><li> Salvar arquivos como imagem pilhas para análise off-line. </li></ol> 5. Automated Ca 2+ Análise Imagem NOTA: É possível visualizar, processar e analisar dados capturados usando muitos pacotes de software comerciais. No entanto, temos desenvolvido um algoritmo para automatizar rapidamente uma identificaçãod análise de Ca 2+ sinais locais. Uma descrição detalhada dos filtros espaciais e temporais utilizados neste algoritmo, a geração dos Af / F 0 e identificação / rotinas de análise pode ser encontrado em 11. Este algoritmo foi desenvolvido para rodar na plataforma Python software open-source, e pode ser obtido, juntamente com a amostra de dados experimentais e instruções detalhadas de usuários, pelo autor correspondente e-mails (ismith@uci.edu). <ol><li> Converta arquivos a serem importados para o algoritmo escrito sob medida para o formato de arquivo multi-plano .tiff. </li><li> Localize a pasta que contém o algoritmo de análise e dê um duplo clique no run.exe. </li><li> Selecione o arquivo .tiff a ser analisado. </li><li> Escolha os parâmetros de análise na caixa de diálogo aberta. Veja a Figura 2A para parâmetros padrão para os dados de exemplo. </li><li> Determinar o nível de preto para ser deslocada da pilha pela imagem seja movendo o cursor para uma parte do campo de visão que n fazot conter uma célula (ver Figura 2B) ou digitando manualmente um nível de preto usando o prompt de &#39;Black Nível Definir&#39;. </li><li> Observe quatro janelas na tela seguinte análise pelo software (Figura 2C-F). C é uma imagem monocromática de descansar fluorescência a partir de células que está sendo analisado. Quadrados brancos sobrepostos na imagem são os locais de evento, determinados como as posições centróide funções de Gauss bidimensionais que equipam os eventos. </li><li> Clique em cada local do evento ou pressione as teclas do cursor para navegar entre os eventos. Ao fazê-lo a subtracção do fundo, Gaussian alisou mudanças de relação de fluorescência (Af / F 0) em cada um desses sites de atualização na janela D. NOTA: Red destacou os acontecimentos estão determinados a se originam de que determinado site e não de &quot;sangrar-through &#39;de atividade localizada para locais adjacentes. O traço azul mais baixo indica que o sinal de fluorescência no local seleccionado excedeu a detecçãolimiar, enquanto que a linha preta identifica eventos provenientes desse local particular (que corresponde ao vermelho eventos realçado). </li><li> Clique sobre um evento vermelho destaque para actualizar a janela E que mostra a evolução temporal do evento, e a janela F, que mostra o perfil espacial do evento, calculados sobre o curso de tempo, em conjunto com o perfil espacial da função de Gauss ajustada. </li><li> Manualmente todos os eventos que foram identificados para que os artefatos podem ser rejeitada de análise. Excluir tais eventos clicando o botão direito no vermelho destacou os acontecimentos. NOTA: Em nossas mãos, fluxo de Ca2 + através dos canais individuais IP 3 R evoca sinais com Af / F 0 cerca de 0,11, então é provável que sejam falsos positivos quaisquer &#39;eventos&#39; detectados sensivelmente menores do que isso. </li><li> Exportar dados Ao selecionar &#39;Salvar em Excel &quot;ou exportação de dados em uma base por célula desenhando em torno da célula de interesse e selecionando&quot; Salvar CellR17 ;. NOTA: Os dados são salvos na mesma pasta que a pilha de imagem original. </li></ol>

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The authors declare that they have no competing financial interests.

Descrevemos aqui protocolos de imagem Ca 2+ eventos locais em células de mamíferos em cultura usando fluorescentes Ca 2+ indicadores. Além disso, descrevemos um algoritmo com uma interface de usuário intuitiva que automatiza a identificação e análise dos dados adquiridos. O procedimento aqui descrito utilizar o Ca 2+ indicador fluorescente Cal-520, mas muitos outros corantes de Ca2 + sensíveis, tais como Fluo-3, Fluo-4, Fluo-8 e Verde Oregon BAPTA-1 desempenho suficien...

Os íons de cálcio (Ca 2+) ubíqua regular uma gama diversificada de processos biológicos, incluindo a expressão do gene, secreção e mudanças de longa duração em plasticidade sináptica 1. Uma maneira através da qual Ca 2+ pode agir de maneira tão diversa é através dos diferentes padrões espaciais e temporais de Ca 2+ sinaliza uma célula pode gerar. Por exemplo, as elevações globais em cytosolic [Ca 2+] contração gatilho no tecido muscular liso 2 Considerando menor, elevações transitórias localizadas (locais Ca 2+ Microdomínios) estimular a expressão essencial para a aprendizagem e memória 3 gene. Citosólico livre [Ca 2+] é mantida a ~ 100 nM em repouso, mas pode levantar-se rapidamente de várias micro-molar seguindo o influxo de Ca2 + no citosol através de canais de Ca2 + de iões -permeable localizados na membrana do plasma e pela a libertação de Ca2 + a partir de s intracelularestores. O nosso laboratório concentra-se o receptor de inositol 1,4,5-trifosfato (IP 3 R), que forma um canal de Ca2 + de libertação localizado na membrana do retículo endoplasmático (ER). Após a ligação de ambos IP3 e Ca 2+ para a activação citosólica sítios do receptor, o canal IP R 3 se abre para libertar o Ca2 + sequestrado dentro do lúmen do ER. A libertação de Ca2 + pode permanecer limitada espacialmente, para um pequeno grupo de IP 3 Rs para gerar um microdomínio citosólica locais de Ca2 + (Ca2 + sopro 4), ou, dependendo da proximidade de aglomerados vizinhos de IP 3 Rs, pode propagar ao longo de uma célula através do recrutamento de vários sites de sopro através de um processo de Ca2 + induzida por Ca2 + -release (CICR) 5,6. A introdução de pequenas molécula fluorescente de Ca 2+ corantes indicadores desenvolvido por Roger Tsien 7, juntamente com o avançado imagi microscopiatécnicas Ng, facilitou muito a nossa compreensão do Ca 2+ sinalização. Recentes avanços em câmeras usadas para microscopia agora permitir transitórias de imagem locais Ca 2+ eventos como puffs com inédita resolução espacial e temporal. Atualmente câmeras EMCCD disponíveis permitem imagem com 128 x 128 pixels em&gt; 500 quadros seg -1 (fps) e da nova geração de complementar semicondutor de óxido metálico (CMOS) câmeras proporcionar maior pixels de resolução e velocidade ainda mais rápido à custa de um pouco maior níveis de ruído. Em conjunto com a microscopia de reflexão interna total (TIRF) agora é possível imagem única Ca 2+ eventos canal 8,9. Esta abordagem permite que a imagem de centenas de canais / eventos simultaneamente, ao gerar grandes conjuntos de dados (cerca de 1 GB por min) que tornam o processamento manual, identificação visual e análise impraticável e colocar um ônus sobre o desenvolvimento de algoritmos automáticos. 2+ sinais locais em células de mamíferos intactos usando fluorescentes Ca 2+ indicadores. Demonstramos ainda um algoritmo desenvolvido no ambiente Python open-source que automatiza a identificação e análise de Ca2 + eventos locais fotografados por ambos TIRF e microscopia de campo largo epi-fluorescência convencional (WF). Embora estas abordagens descrevemos no contexto da PI 3 -generated Ca 2+ sinais, eles são facilmente recuperáveis ​​para estudar alterações locais na citosólica [Ca 2+] emanados a partir de uma variedade de canais de Ca2 + de iões -permeable localizados quer na superfície membrana ou organelas intracelulares 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

Citossólica Ca 2+ regular vários aspectos da atividade celular em quase todos os tipos de células, processos tão diversos como a transcrição de genes, excitabilidade elétrica e proliferação celular controle. A diversidade e especificidade de Ca 2+ sinalização deriva de mecanismos pelos quais Ca 2+ sinais são gerados para atuar em diferentes escalas de tempo e espaciais, que vão desde oscilações e ondas em toda a célula que ocorrem durante os períodos de minutos locais transitórias Ca 2+ microdomínios (Ca 2+ puffs) milésimos duradoura. Os recentes avanços na eletrônica multiplicado CCD (EMCCD) câmeras permitem agora para a imagem latente de 2+ sinais locais de Ca com uma resolução espacial de 128 x 128 pixels em taxas de quadros&gt; 500 s-1 (FPS). Esta abordagem é altamente paralelo e permite a monitorização simultânea das centenas de canais ou sítios sopro numa única experiência. No entanto, a grande quantidade de dados gerados (cerca de 1 Gb per min) tornar a identificação visual e análise de Ca 2+ eventos locais impraticável. Aqui nós descrevemos e demonstrar os procedimentos para a aquisição, detecção e análise de IP local 3 mediada por Ca 2+ sinais em células de mamíferos intactos carregados com Ca2 + indicadores que utilizam tanto de campo amplo epi-fluorescência (WF) e de reflexão interna total fluorescência (TIRF) microscopia. Além disso, descrevemos um algoritmo desenvolvido dentro do open-source software ambiente Python que automatiza a identificação e análise desses Ca 2+ sinais locais. O algoritmo localiza locais de Ca 2+ liberação com resolução sub-pixel; permite a revisão dos dados do usuário; e produz sequências de tempo de sinais de relação de fluorescência em conjunto com amplitude e dados cinéticos em uma tabela do Excel-compatível.

Figura 1A mostra uma imagem WF de descansar Cal-520 fluorescência em células neuroblastoma SH-SY5Y humanos também carregados com ci-IP 3. A exposição destas células para um flash de 100 ms para UV foto-release i-IP 3 suscitou transientes de Ca2 + puffs em locais discretos (apontadas pelos círculos brancos na Figura 1A). Vestígios de fluorescência medidos nesses locais mostraram uma fase crescente, devido à rápida aberturas transientes de IP <s...

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.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

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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Imaging Local Ca2+ Signals in Cultured Mammalian Cells

10.7K visualizações.  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.

Vídeo: 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.

Longo prazo células de mamíferos utilizando imagens do Largo-Campo Microscopia

Biologia

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.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

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.

EsiRNA MISSÃO para a seleção RNAi em células de mamíferos

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.

Fluorescência baseada em Medição da loja operada entrada de cálcio em células vivas: a partir de células cancerosas cultivadas em fibra muscular esquelética

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.

Análise da posição do ciclo celular em células de mamíferos

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.

Visualização do retículo endoplasmático Subdomains em células cultivadas

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.

Automated Análise de Ca dinâmico<sup&gt; 2 +</sup&gt; Sinais em seqüências de imagens

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.

Screening siRNA identificar Ubiquitin e ubiquitina-como reguladores do sistema de caminhos biológicos em células de mamíferos cultivadas

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.

Medição dos Níveis de heme em células de mamíferos Síntese

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.