Produzido por Loredana Manfra do Instituto para a proteção ambiental e à pesquisa (ISPRA-Departamento de Monitoramento da Qualidade Ambiental) e Savorelli Federica da Agência Regional de Proteção Ambiental (Emilia-Romagna, Itália). Os membros de referência para o subgrupo de trabalho sobre o crustáceo Artemia dentro do Grupo métodos biológicos - água salgada / água salobra e Sedimentos da Comissão UNICHIM da Qualidade da Água (Instituto para a operação de normalização técnica no sector químico). Videografia e edição por Marco Pisapia de ISPRA (Unidade de Web). Coordenação de Produção por Anna Maria Cícero e Magaletti Erika de ISPRA (Departamento de Monitoramento da Qualidade Ambiental). Gostaríamos de agradecer à Agência Regional de Proteção Ambiental da Emilia-Romagna, Ferrara Branch, o Grupo de Métodos Biológicos - água salgada / salobraÁgua e Sedimentos da Comissão UNICHIM sobre Qualidade da Água e Migliore Luciana, da Universidade Tor Vergata (Roma) da sua cooperação; Boscolo Rossella e Massimo Gabellini de ISPRA (Departamento de Prevenção e Mitigação de Impactos) pelo apoio financeiro; Matassa Fabio e Londres Escola de Idiomas em Roma, pelo seu apoio linguístico.

1. Método Padrão O nosso teste envolve a exposição larvas de Artemia a um intervalo de concentrações da substância ou uma amostra de ensaio para a finalidade de determinar os níveis de concentração ou diluição que poderiam causar a morte de 50% dos organismos (CL 50) expostos ao longo de 14 dias e que satisfaça as condições definidas por este método. Se necessário e possível, a seguinte pode também ser determinada: a) o nível de concentração que causa a morte de 20% dos organismos expostos (LC 20), b) o nível de concentração mais elevada analisados ​​que não determina uma maior taxa de mortalidade do que a do o controlo negativo (NOEC); a concentração mais baixa testada que determina, após 14 dias, uma maior taxa de mortalidade do que a do controle negativo (LOEC). 2. Testing Materials 2.1 ovos de Artemia Para realizar testes, Artemia franciscana </em&gt; Larvas de menos de 48 horas de idade (náuplios) são necessários, no estádio II ou III, obtida a partir de ovos recentemente chocadas 5. Ovos ou cistos certificados utilizados para apoiar os testes estão disponíveis comercialmente, por exemplo, da Divisão de Pesquisa de Qualidade, EUA Agência de Proteção Ambiental, Cincinnati OH 45268, EUA ou do Laboratório de Pesquisas Biológicas da poluição aquática, Universidade de Ghent, na Bélgica. 2,2 diluição Água Água salgada sintética pode ser usado como água de diluição, preparada por dissolução de reconhecidos reagentes de qualidade analítica ou uma formulação comercialmente disponível em água destilada ou desionizada. Misturas de sal para a criação do ideal de água salgada estão comercialmente disponíveis, por exemplo, as misturas Instant Ocean. Recomenda-se uma água de diluição, com uma salinidade igual a 35 (± 2) PSU, bem como uma concentração de oxigénio dissolvido superior a 80% do valor de saturação e uma temperatura de 25 (± 2) egraus; C. Após arejamento durante 48 horas e de filtração em filtros com uma porosidade de pelo menos 0,45, a diluição pode ser armazenado durante um máximo de 30 dias no escuro a uma temperatura de 0-4 ° C. 2,3 cultura de algas Recomenda-se que os organismos de teste são alimentam de microalgas Dunaliella tertiolecta, que crescem exponencialmente e atingir densidades de 1.3x10 6 - 2.0x10 6 células / ml. Testes em amostras ambientais requer culturas com densidades superiores a 2.0x10 6 células / ml 6. Sugere-se que Dunaliella tertiolecta cresce em meio de cultura contendo água do mar (30 (± 2) PSU e 20 (± 1) ° C) com a adição de nutrientes essenciais, minerais e vitaminas. Algumas indicações sobre a composição do meio de algas e preparação são apresentados no Apêndice. A densidade de células inicial é sugerido para ser de aproximadamente 10 5 células / ml. A cultura de algas é mantida numatermostato ambiente a 20 (± 1) ° C, iluminada com lâmpadas fluorescentes 3000 (± 300) lx, com um rácio de fotoperíodo de 16 horas de luz e 8 horas de escuridão. Espécies de fitoplâncton Outros também poderia ser utilizado para a alimentação mas seria necessário fazer estudos comparativos com estas espécies de fitoplâncton para encontrar os &quot;equivalentes&quot; espécies a Dunaliella tertiolecta, porque as condições experimentais (por exemplo, densidades celulares iniciais, de iluminação com luz, temperatura, composição do meios de cultura e tempo de exposição) varia entre as espécies do fitoplâncton. Além da tolerância do microalgas para diferentes poluentes precisa ser considerado, porque os efeitos de poluentes sobre o crescimento e desenvolvimento de impacto fitoplâncton também para a Artemia. 2,4 substância de referência A substância de referência no presente ensaio é dodecil sulfato de sódio. 2,5 de vidro para laboratório <ol><li> Recipientes de teste, incluindo 100 ml provetas de vidro de borossilicato (18 para executar os testes e 18 para a transferência de soluções). </li><li> Um balão (por exemplo, frasco de 500 ml) para preparar a solução padrão. </li><li> Seis frascos (por exemplo, 200 frascos ml) para preparar as soluções de teste. </li><li> De vidro ou poliestireno 5 cm de diâmetro pratos de Petri com tampas removíveis para ser usado para activar os cistos e transferindo Artemia a partir do prato incubação para os recipientes de teste. </li><li> Pipetas de vidro Pasteur (com pontas arredondadas por chama) para a transferência de náuplios. </li><li> Vidro cânulas ou Pasteur 3 ml descartáveis ​​pipetas de plástico (a ser cortado) para a transferência de Artemia. </li><li> Micropipetas e pipetas graduadas para preparar as soluções de teste. </li><li> Seis cilindros graduadas (por exemplo, 50 cilindros ml) para a transferência de soluções de teste a partir dos frascos para tele testando recipientes. </li></ol> 3. Ativação de cisto 3,1. Placas de Petri contendo cistos + de derramar água Para gerar organismos de teste, 20 mg de cistos são colocados em uma placa de Petri contendo 12 ml de água de diluição 48 horas antes do teste. 3,2. De incubação na luz e no escuro O prato Petri é mantida a 25 (± 2) ° C e à 4000 (± 1000) lux (lúmens por metro quadrado) a intensidade da luz durante uma hora. 3,3. Substituição do meio usando o microscópio Após 24 horas, a incubação de larvas são transferidos para uma placa de Petri de novo cheio com água artificial. A transferência é realizada utilizando o microscópio, ou seja, usando uma fonte de luz de modo que a incubação de larvas, phototropic migram para o feixe de luz. Uma pipeta de Pasteur de vidro é usado para fazer a transferência, assegurando que apenas o nascente larvas are transferido e não os quistos ou as larvas ainda em membranas. 3,4. A incubação no escuro O prato contendo as larvas é colocado numa câmara termostática escuro durante 24 horas a 25 (± 2) ° C. 4. Preparação das soluções de teste 4,1. Pesando a substância + transferir para o balão Recomenda-se que a solução padrão para a substância de teste é preparado por dissolução de 0,5 g da substância em um balão de 500 ml. O balão é cheio com água desionizada ou destilada ea solução é agitada até que a substância de teste é completamente dissolvido. Soluções têm de ser preparadas no momento da utilização, salvo se não se sabe se a substância é estável em solução. Nesse caso, a solução padrão podem ser preparados até dois dias antes do teste. 4,2. Preparação do controlo negativo, adicionando a água de diluição e algas with a 10 ml de pipeta O controlo negativo é preparada em um balão (por exemplo, o volume de 200 ml) por adição de uma alíquota da suspensão de algas para a água de diluição de modo que uma densidade de 10 5 células / ml é obtido. 4,3. Preparar as soluções de testes: a adição de água de diluição, as algas e uma solução padrão Sugere-se que as soluções de teste são preparados em cinco balões de 200 ml por adição da solução padrão para a água de diluição nas quantidades especificadas de modo a que as concentrações desejadas para teste é obtido. Para alimentar os organismos, alíquotas de uma suspensão de microalgas Dunaliella tertiolecta pode ser utilizado e adicionado a atingir uma densidade de 10 5 células / ml quando as soluções de ensaio são preparados. A seguinte ordem de adição é recomendada: água de diluição, a suspensão de algas e solução padrão. Após a preparação das soluções de teste, eles têm de ser utilizados no ensaio o mais rapidamente possível. 4,4. Transferir a partir de soluções o balão para os recipientes de teste, em seguida, para as placas de Petri Quantidades iguais (50 ml) das soluções de ensaio são introduzidos nos recipientes de ensaio, utilizando o cilindro graduado. Três réplicas para cada concentração são feitas. Para cada série de testes, um recipiente de controle é preparado com uma quantidade de água de diluição igual ao volume das soluções de ensaio. Volumes iguais (cerca de 12 ml) de as soluções de ensaio são introduzidos nas placas de Petri. Para cada série de testes, um prato de Petri de controlo é usado. 5. Preparação para Testes 5,1. Náuplios na fase II-III Quarenta e oito horas após a activação do cisto, os náuplios atingir a II-III fase larval e são, em seguida, utilizável para o teste. 5,2. Náuplios ser transferido para as placas de Petri, usando o microscópio A placa de Petri utilizado para cisto<html> activação é levada para fora da câmara termostática. Uma pequena quantidade de larvas são transferidas para as placas de Petri contendo o controlo e as soluções de teste. Esta tarefa é para ser realizado com o microscópio utilizando uma pipeta de diâmetro suficientemente larga de modo que os organismos não ficar danificado. Nesta fase do ensaio, a transferência pode ser realizada com uma pipeta de Pasteur de vidro (com ponta arredondada à chama) e uma fonte de luz posicionada lateralmente que encoraja a Artemia a agregação. 5,3. Náuplios transferência a partir das placas de Petri em recipientes de teste com o microscópio Dez larvas são transferidas a partir dos pratos de Petri de ensaio soluções para os recipientes de teste. Também esta operação tem que ser executada, utilizando a pipeta de Pasteur e microscópio. Recomenda-se que um volume não superior a 1 mL é transferido durante a passagem das larvas para não afectar o volume global de sistema de teste. e_step &quot;&gt; 5.4 Os recipientes cheios cobertos com parafilme As provetas de ensaio são cobertos com parafilme (deixando um espaço para a passagem de ar), e mantido a uma temperatura de 25 (± 2) ° C durante toda a duração do teste e com uma iluminação de 900 (± 100) lx com um fotoperíodo proporção de 14 horas de luz e 10 horas de escuridão. 6. Substituindo o suplemento Médio e Alimentação 6,1. Controle da taxa de sobrevivência Dois dias após o teste foi iniciado, e, em seguida, após dias 5, 7, 9 e 12, a Artemia é observada ao microscópio a fim de verificar a taxa de sobrevivência e para substituir o meio eo suplemento alimentar. Número de organismos vivos é contado em cada contêiner de teste. Após a observação ao microscópio e estimulação mecânica leve (por exemplo, tocar as larvas com uma pipeta Pasteur de vidro) organismos que não apresentam algum movimento por cerca de 10 segundos deve ser considerado morto. 6,2. Preparação de uma solução de teste exibindo amostragem algas com uma pipeta de 10 ml e amostragem da substância a ser testada com uma micropipeta. Transferir a partir de soluções o balão com os recipientes de teste Embora o teste, as soluções de teste devem ser periodicamente substituído, e preparado no mesmo dia em que eles são para ser usado. As soluções de ensaio são feitos a partir da solução padrão previamente preparada. 6,3. Transferência de Artemia, utilizando uma pipeta de Pasteur de corte, a partir dos recipientes de teste antigos para os três recipientes novos A transferência de Artemia é realizada usando uma pipeta com um diâmetro suficientemente larga de modo a não danificar os organismos. Durante esta fase, de 3 ml de plástico pipeta de Pasteur (a ser cortado) pode ser utilizado. 6,4. As larvas num copo contendo quer Artemia vivo ou morto Após 1<html> 4 dias de exposição no final do teste, o número de sobreviventes larvas é contado e registado na folha de cálculo. LC 50 e / ou LC 20, NOEC e LOEC menos 14 dias, são calculados e registrados, bem como limites de confiança de 95% no caso de métodos apropriados, e de cálculo. 7. Os resultados representativos No final do ensaio (14 dias), calcular a percentagem de mortalidade para cada concentração, de acordo com a seguinte fórmula: (M s / N) * 100 Onde: M s é o número médio de indivíduos morreram na amostra analisada N é o número total de indivíduos expostos Tabela 1 relata um exemplo de dados de um ensaio de (14d) com Artemia expostos a dodecil sulfato de sódio. <table border="1"><tbody><tr><td> Concentrações (mg / l) </td>&quot;&gt; Número de pessoas morreram em cada réplica </td><td> Mortalidade (%) </td></tr><tr><td></td><td> Eu </td><td> II </td><td> III </td><td></td></tr><tr><td> Controle negativo </td><td> 0 </td><td> 2 </td><td> 1 </td><td> 10 </td></tr><tr><td> 1,56 </td><td> 1 </td><td> 2 </td><td> 0 </td><td> 10 </td></tr><tr><td> 3,12 </td><td> 2 </td><td> 0 </td><td> 2 </td><td> 13 </td></tr><tr><td> 6,25 </td><td> 3 </td><td> 2 </td><td> 3 </td><td> 27 </td></tr><tr><td> 12,5 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 90 </td></tr><tr><td> 25,0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr><tr><td> 50,0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr></tbody></table> Além disso, determinar oLC valor 50 por um lote Gaussian gráfica logarítmica ou utilizando métodos estatísticos adequados (por exemplo Spearman-Karber ou Método Probit). O cálculo da LC 50 para os dados da tabela 1 é 8,18 (7,15-9,37) mg / l. Se a concentração máxima testada causou uma menor taxa de mortalidade de 50%, você não deve continuar para o cálculo do 50 LC valor que seria, então, não confiáveis ​​ou até mesmo não-computável. Neste caso, no entanto, seria apropriado para repetir o ensaio, alargando a gama de concentrações testadas. Alternativamente, o LC 50 valor é mais correctamente expresso como maior do que a maior concentração testada, possivelmente indicando a percentagem de mortalidade na maior concentração testada ea concentração mais elevada que corresponde a uma nenhum efeito. Os resultados são considerados válidos se no fim de testar as condições a seguir foram atendidos: <ol class="lalpha"><li> a taxa de controle de mortalidade médiaé ≤ 20%; </li><li> onde usando dodecil sulfato de sódio, o LC 50, 14 dias está incluído no 8,0 (± 5) mg / l de intervalo. </li></ol> Se as condições acima não foram cumpridos, todos os dados obtidos com o mesmo lote de organismos devem ser considerados inválidos e repetidos testes.

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Artemia é um dos organismos de teste mais valiosos disponíveis para testes de ecotoxicidade e de investigação feito até agora permite-nos afirmar que é possível manter várias opções relacionadas com Artemia uso em Toxicologia e Ecotoxicologia. As características que tornam este organismo em uma espécie com potencial para bioensaios são: uma ampla distribuição geográfica, alta adaptabilidade às condições ambientais adversas e nutrientes variados, cultura laboratorial relativamente sim...

Produzido por Loredana Manfra do Instituto para a proteção ambiental e à pesquisa (ISPRA-Departamento de Monitoramento da Qualidade Ambiental) e Savorelli Federica da Agência Regional de Proteção Ambiental (Emilia-Romagna, Itália). Os membros de referência para o subgrupo de trabalho sobre o crustáceo Artemia dentro do Grupo métodos biológicos - água salgada / água salobra e Sedimentos da Comissão UNICHIM da Qualidade da Água (Instituto para a operação de normalização técnica no sector químico). Videografia e edição por Marco Pisapia de ISPRA (Scientific Advisory e Serviços de Documentação). Coordenação de Produção por Anna Maria Cícero e Magaletti Erika de ISPRA (Departamento de Monitoramento da Qualidade Ambiental). Gostaríamos de agradecer à Agência Regional de Proteção Ambiental da Emilia-Romagna, Ferrara Branch e BiologGrupo de Métodos ical - água salgada / água salobra e Sedimentos da Comissão UNICHIM da Qualidade da Água da sua cooperação; Rossella Boscolo e Massimo Gabellini de ISPRA (Departamento de Prevenção e Mitigação de Impactos) pelo apoio financeiro, e da London School of Languages, em Roma por seu apoio linguístico. <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número do produto </td></tr><tr><td> Dodecil sulfato de sódio </td><td> Sigma-Aldrich </td><td> L4390 </td></tr></tbody></table> Apêndice Os meios de comunicação de algas tem de ser preparada utilizando reagentes de qualidade analítica. Um litro de solução nutritiva é preparada por dissolução de 300 g de NaNO 3, 30 g de KH g PO 4 e 20 2 de NH4CI em água destilada. Quatro soluções iniciais (A, B, C, D) De minerais são preparados como se segue: <ul><li> Um litro de solução &quot;A&quot; contendo 30 g de ZnSO 4 H2O, 25 g de CuSO4 5 H2O, 30 g de COSO 4 7 H2O e 20 g de MnSO4 H2O em água destilada; </li><li> Um litro de solução &quot;B&quot; contendo 50 g de FeCl 3 • 6 H2O em água destilada; </li><li> Um litro de solução &quot;C&quot;, contendo 25 g de Na 2 MoO 4 • 2 H 2 O em água destilada; </li><li> Um litro de solução &quot;D&quot;, contendo 50 g di Na 2 EDTA • 2 H 2 O em água destilada. </li></ul> Um litro de uma solução final de minerais (solução &quot;E&quot;) é obtida adicionando alíquotas das soluções acima mencionadas para a água destilada. A seguinte ordem de adição é recomendado: 100 ml de solução &quot;D&quot;, 10 ml de solução &quot;C&quot;, 10 ml de solução &quot;B&quot; e 10 ml de solução &quot;A&quot;. Três iniciativasl soluções (F, G, H) de vitaminas são preparados como se segue: <ul><li> Um litro de solução &quot;F&quot; contendo 100 mg de biotina em água destilada; </li><li> Um litro de solução de &quot;G&quot; contendo 10 g de tiamina em água destilada; </li><li> Um litro de solução &quot;H&quot; contendo 100 mg de cobalamina em água destilada. </li></ul> Um litro de uma solução final de vitaminas (solução &quot;I&quot;) é obtida adicionando-se 10 ml das três soluções iniciais para a água destilada. Um litro de meio de algas é preparado por dissolução de 1 ml da solução de nutrientes, 1 ml da solução de mineral final (solução &quot;E&quot;) e 1 ml da solução de vitamina final (solução &quot;I&quot;) na água do mar. Este último pode ser obtida por dissolução reconhecidos reagentes de qualidade analítica ou uma formulação comercialmente disponível em água destilada ou desionizada. Misturas de sal para a criação do ideal de água salgada estão comercialmente disponíveis, por exemplo, as misturas Instant Ocean. É recomended a utilização de uma água do mar com salinidade de 30 (± 2) PSU e temperatura de 20 (± 1) ° C.

long-term lethal toxicity test with the crustacean artemia franciscana

Nossas atividades de pesquisa sobre a utilização de métodos biológicos para a avaliação da qualidade ambiental, com particular referência à água salgada / água salobra e sedimentos. A escolha dos indicadores biológicos devem ser baseadas em conhecimentos científicos confiáveis ​​e, possivelmente, sobre a disponibilidade de procedimentos padronizados. Neste artigo, apresentamos um protocolo padronizado que utilizou o crustáceo marinho Artemia para avaliar a toxicidade de produtos químicos e / ou marinhos matrizes ambientais. Os cientistas propõem que o camarão (Artemia) é um candidato adequado para o desenvolvimento de um bioensaio padrão para utilização em todo o mundo. Uma série de artigos têm sido publicados sobre os efeitos tóxicos de vários produtos químicos e substâncias tóxicas sobre Artemia salina (Artemia). A principal vantagem do presente crustáceo para estudos de toxicidade é a disponibilidade geral dos cistos secos; estes podem ser imediatamente utilizado no teste e de difícil cultivo não é exigido <sup&gt; 1,2. Cisto ensaios baseados toxicidade são baratos, continuamente disponível, simples e confiável, e são, portanto, uma resposta importante para as necessidades de rotina de triagem de toxicidade, por exigências de controlo industriais ou para fins de regulamentação 3. O método proposto envolve a mortalidade como um ponto final. O número de sobreviventes foram contadas ea percentagem de mortes foram calculados. As larvas foram considerados mortos se eles não apresentaram qualquer movimento interno ou externo durante vários segundos de observação 4. Este procedimento foi testes padronizados uma substância de referência (dodecil sulfato de sódio); alguns resultados são reportados neste trabalho. Este artigo acompanha um vídeo que descreve o desempenho de testes de toxicidade processual, mostrando todos os passos relacionados com o protocolo.

Watch this Scientific Journal Video about Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana at JoVE.com

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Este estudo diz respeito ao desenvolvimento e padronização de um protocolo metodológico valioso para determinar toxicidade a longo prazo (14 dias) letal exercida por substâncias químicas, efluentes industriais ou esgotos e líquidos amostras ambientais sobre o crustáceo de água salgada,<em&gt; Artemia franciscana</em&gt;.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

long-term-lethal-toxicity-test-with-the-crustacean-artemia-franciscana

Research

JoVE Journal

Biology

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

19.5K visualizações.  Institute for Environmental Protection and Research. Este estudo diz respeito ao desenvolvimento e padronização de um protocolo metodológico valioso para determinar toxicidade a longo prazo (14 dias) letal exercida por substâncias químicas, efluentes industriais ou esgotos e líquidos amostras ambientais sobre o crustáceo de água salgada, Artemia franciscana.

Vídeo: Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Longo prazo Cultura de Amostras de mama Humano do Câncer e sua análise por meio de tomografia óptica de Projeção

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Longo prazo, de alta resolução de imagem Lapse Confocal Tempo de<em&gt; Cotilédone Arabidopsis</em&gt; Epiderme durante a germinação

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enriquecimento de série-tronco de esperma e células progenitoras (SSC) em Cultura para Derivação de longo prazo Adultos Linhas rato SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

Ablação de uma única célula de oito células de embriões de Crustáceo anfípodes<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicamadas de montagem para longo prazo Microscopia de Luz Folha de Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA, um método para montagem de larva drosophila para imagem de lapso de tempo de longo prazo

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Isolamento, Transfecção e Cultura de Longo Prazo de Cardiomiócitos de Ratos Adultos e Ratos

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Aplicação do movimento passivo da cabeça para gerar acelerações definidas nas cabeças dos roedores

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Coleta e Manutenção a Longo Prazo de Formigas Cortadeiras (Atta) em Condições de Laboratório

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...