Os autores gostariam de agradecer ao Dr. Leonid Volkov e Éric Bouchard para a sua assessoria técnica em relação a microscopia de epifluorescência. Ajuda financeira Financiado pelos Institutos Canadenses de Pesquisa em Saúde (concessão # MOP-93781) para GB

Cuidados com os animais foi em conformidade com o cuidado dos animais e uso comissão Université de Sherbrooke. 1. Tubo de Preparação <ol><li> O dia antes da separação de células, adicionar 1-2 mL de soro bovino fetal inactivado pelo calor (FBS) a 5 ml de polipropileno tubos de fundo redondo, e a 15 ml e 50 ml de polipropileno tubos cónicos. Passo crítico: Certifique-se de que todos os tubos usados ​​no protocolo é revestido. Nota: revestimento FBS impede que as células germinativas de furar a parede do tubo. </li><li> Lentamente misturar O / N por inversão a 4 ° C para revestir uniformemente os tubos utilizando um rotor. </li><li> No dia seguinte, retire FBS a partir de tubos revestidos por decantação. <ol><li> * Passo crítico: Certifique-se de que os 5 ml de polipropileno tubos de fundo redondo e os 15 e 50 ml tubos utilizados para preparação de células (protocolo passos 2,1-2,11) estão completamente esvaziados de FBS para impedir Fanning (ou seja, desvio impreciso dos fluxos laterais). Usar uma pipeta de P200 para remover o FBS. <br/&gt; Nota: Residual FBS em tubos antes da classificação pode levar a uma perda de células ou contaminação de outros tubos utilizados durante a separação de células, </li><li> Deixar um pequeno volume residual de FBS (cerca de 100-200 mL) no 5 ml de polipropileno rodada tubos de fundo utilizados para a recolha de células purificadas. </li></ol></li></ol> 2. Preparação celular <ol><li> Sacrificar um rato macho sob anestesia com O2 / isoflurano (indução 5%, em seguida, mantida a 2%), seguida por asfixia com CO2. </li><li> Especial e do decapsulate ambos os testículos e mediu com pequenas tesouras em 500 ul de tampão de triagem para um tubo de 1,5 ml. </li><li> Lavar as células germinativas de túbulos seminíferos por gentil pipetagem cima e para baixo no tubo de 1,5 ml, com um primeiro truncado 100-1.000 ul ponta, e depois com uma ponta intacta 100-1.000 ul. </li><li> Remover detritos e aglomerados por uma etapa de filtração usando um filtro de células 40 pm e recolher filtrado para 50 ml prev tubo cônicoiously revestido com FBS no passo 1. </li><li> Lavar o filtro uma vez com tampão de triagem para um volume total de 3 ml e transferência do filtrado em tubo de 15 ml previamente revestida na etapa 1. </li><li> Adicionar 16 ul de EDTA 50 mM, pH 8 por mililitro de suspensão de células (48 uL de 3 ml) de modo a obter uma concentração final de EDTA a 0,8 mM. </li><li> Para fixar as células germinativas, adiciona-se lentamente 3 volumes de etanol gelado a 100%, utilizando uma pipeta de 10 ml com agitação suave (vórtice a baixa velocidade), a qual irá produzir uma suspensão leitosa. Passo crítico: adição lenta de etanol é importante e crucial para preservar a integridade da preparação de células. Notamos que a rápida adição de etanol provoca a lise celular, resultando em uma importante redução de triagem eficiência. Para obter uma suspensão de células de 3 ml, 9 ml de etanol deve ser adicionado em cerca de 1 min. </li><li> Incubar as células em gelo durante 15 min, com mistura ocasional por inversão. </li><li> Centrifugar a suspensão de células a 800 xg durante 5 min e remover a clarasobrenadante. </li><li> Suavemente ressuspender as células germinativas fixas em 2 ml de tampão de triagem. </li><li> Adicionar 4 ul de corante de ADN SYTO16 (solução 1 mM em DMSO) e incuba-se durante pelo menos 30 min (protegida da luz). Nota: Os melhores resultados são obtidos usando triagem SYTO16 uma vez que é um corante de ADN permeável que é facilmente incorporado nos núcleos altamente compactadas de espermatídeos. </li></ol> Sorter 3. celular Set Up Nota: Aqui, a 4 de laser (405 nm - violeta, 488 nm - azul, 561 nm - verde-amarelo, 633 nm - vermelho) 20 parâmetro de separação de células BDFACSAria III é usado. O software BD FACSDiva 6.1.3 é usado para visualizar e analisar os dados. As configurações podem variar dependendo do tipo de classificador utilizado. <ol><li> Esteriliza-se o classificador de FACS através da execução de etanol a 70% como fluido de revestimento durante o processo de encerramento de fluidos (menu &quot;Citómetro&quot; no software) antes da triagem. Selecione &quot;Cytometer&quot; na barra de ferramentas do software e escolha &quot;sh Fluidicutdown &quot;. Siga as etapas mencionadas pelos processos. </li><li> Preparar e filtrar 1x PBS com um filtro de 0,22 um, de modo a eliminar quaisquer cristais e contaminantes. Encher o reservatório de bainha para a linha de soldadura superior no interior do tanque. </li><li> Inicie o software e fazer o login. Comece o classificador FACS para aquecer os lasers durante pelo menos 30 minutos antes da classificação. Executar dois processos de inicialização fluídicos para lavar o etanol a partir dos fluidos. Selecione &quot;Cytometer&quot; na barra de ferramentas do software e escolha &quot;startup Fluidic&quot;. Siga os passos mencionados pelos processos. Nota: Isso restaura fluídica com fluido de revestimento normal (1x PBS). </li><li> Limpe o bloco de triagem e as placas de deflexão com água destilada e seque-as completamente. Sonicar um bico de 100 | iM colocada num tubo de água destilada durante 2 minutos num banho de ultra-sons. Limpe o bico minuciosamente. </li><li> Inserir o bocal 100 fim na célula de fluxo e virar a alavanca de bloqueio dos ponteiros do relógio para o bocal 12posição h. Defina a pressão do invólucro a 20 psi. </li><li> Ligue a corrente e ajustar a amplitude de obter valores-alvo para a freqüência (cerca de 30), queda de 1 (cerca de 150) e Gap (cerca de 12) e para estabelecer um padrão de gotículas breakoff estável (poucas gotas por satélite). Desligue atenuação. </li><li> Certifique-se de que o fluxo é linear e atinge o centro do aspirador de resíduos, alterando o ângulo do tipo bloco. Nota: Um bocal 130 uM a 10 psi também foi testado e nenhuma diferença óbvia foi encontrado. </li><li> Definir atraso laser para 0 para o -77,39 azul, para o vermelho, 37,33 para o violeta e -39,85 para os lasers verde-amarelo. </li><li> Definir áreas de escala para 1.14 para o 1.0 azul, para o vermelho, 0,75 para o violeta e 0,96 para os lasers verde-amarelo. </li><li> Usando um Sort Test (na &quot;Side Stream&quot; janela do software), certifique-se que os fluxos secundários não são abanando. Na janela &quot;Side Stream&quot;, ajuste os controles deslizantes de tensão de cada fluxo de lado para que eles atingiram o middlE de tubo de colheita de teste. Se o fluxo de centro não é apertado, ajustar o 2º, 3º e 4 configurações th gota para restringir o fluxo de centro. </li><li> Inicie o Setup Cytometer e Rastreamento (CS &amp; T) na aplicação de software (menu &quot;Cytometer&quot;). </li><li> Use a configuração e acompanhamento de contas de citómetro a 1 gota por cada 350 mL de automatizar a caracterização e monitoramento do desempenho citômetro utilizando as instruções do fabricante. </li><li> Sair da aplicação CS &amp; T. Aplicar configuração CS &amp; T, certifique-se os parâmetros de transmissão ainda permitir um padrão de gotículas breakoff estável e ligue o botão Sweet Spot conforme descrito nas instruções do fabricante (na &quot;janela breakoff&quot;). </li><li> Otimizar o valor Gota Delay utilizando partículas fluorescentes (ver Tabela de Materiais). <ol><li> Instale os tubos de coleta no suporte. Abra a porta do bloco de classificação. Na janela &quot;Side Stream&quot;, ligue tensão, em seguida, selecione &quot;Teste de tipo &quot;e clique no botão Aspirador gaveta para abrir a gaveta e ter acesso aos tubos de coleta. </li><li> Otimizar os fluxos laterais de forma que eles vão para o centro do tubo. Ajustar os valores e controles deslizantes conforme necessário. </li><li> Feche a porta do bloco de classificação e certifique-se de ajustar o relógio comparador de obter o ponto mais brilhante pérola no centro. Gaveta resíduos Unselect, uma espécie de ensaio e de tensão. Retire os tubos de coleta. </li><li> Optimizar a gota atraso de acordo com as instruções dos fabricantes. <ol><li> Resumidamente, agitar vigorosamente frasco grânulo e diluir 1 gota de contas em 0,5 ml de PBS. </li><li> Usando as instruções dos fabricantes, definir o atraso gota usando o modelo experimento Gota Delay disponíveis no software. Como alternativa, use o recurso Auto Gota Delay para definir o atraso queda. </li><li> Coloque o tubo de partículas fluorescentes e ajustar a taxa de fluxo até que a taxa limite é de aproximadamente 3.000 a 4.000 eventos / segundo. Defina o tipo de precisão &quot;inicial&quot; e últimaly &quot;afinar&quot; na janela &quot;Ordenar disposição&quot;. Selecione o filtro óptico e classificar esferas. Ajustar a gota Atraso até ~ 100% são classificados para a esquerda. Nota: Este passo é crítico para certificar-se que as células de interesse espécie numa corrente de lado. Os grânulos e o filtro óptico são usados ​​para alcançar perto de um desvio de gotículas de 100%. </li></ol></li></ol></li><li> Coloque um ND (densidade neutra) filtro FSC 1.5 na extremidade esquerda do bloco detector FSC. Defina a extensão de janela para 2,00 ms e a escala FSC Área de 1,00 (no separador &quot;Laser&quot; na janela &quot;Cytometer&quot;). </li><li> Antes de carregar o tubo de ensaio, colocar um filtro de linha de amostra de 50 um na extremidade da linha de amostra para evitar a aglomeração. Para fazer isso, selecione &quot;Alterar Filtro de exemplo&quot; no menu &quot;Cytometer&quot;. </li><li> Teste a composição da amostra de fluido para alcançar uma boa separação sem abanando entre fluxo de lado e para impedir as células de degola para a Polipropiletubos ne. <ol><li> Coloque o tubo de amostra, ative as placas de deflexão e selecione &quot;Test tipo&quot; com a gaveta fechada. Olhe para fluxos secundários se a separação ocorre sem muito Fanning. Nota: A ventilação é provavelmente devido a uma elevada concentração de proteínas no tampão de amostra. </li></ol></li><li> Coloque o tubo de amostra no instrumento FACS. Seleccionar uma amostra de agitação de 300 rpm e uma temperatura de amostra de 4 ° C. Selecione &quot;Obter dados&quot; no &quot;Aquisição Dashboard&quot;. </li><li> Se necessário, diluir a amostra para alcançar uma taxa de eventos apropriado para um máximo de 5000 eventos / s (a uma taxa de fluxo máxima de 3.0). Estritamente respeitar estes limites de modo a manter a pureza das células separadas. </li><li> Para analisar e classificar as células, otimizar o valor limite FSC para eliminar detritos, sem interferir com a população de interesse. Em escala logarítmica, ajustar a tensão do detector de tubo foto-multiplicador (PMT), com o filtro de 530/30, a fim de fazer-se que o sinal de fluorescência da população total se encaixa dentro da escala, e portão na população positiva (Figura 1, porta 1). </li><li> Produzir um Scatter-área Forward (FSC-A) área-Side Scatter / (SSC-A) parcela da população positiva SYTO16 e ajustar o FSC-A para cerca de 110 V na escala linear, eo SSC-A para cerca de 150 V em escala logarítmica para visualizar todos os eventos enquanto excluindo muito grandes células e agregados celulares. </li><li> Na janela &quot;Ordenar Layout&quot;, defina o &quot;Classificar Precision Mode&quot; para &quot;pureza 4-way&quot; (máscara Rendimento: 0, Máscara Pureza: 32, máscara de fase: 0). Selecione &quot;Continuous&quot; no menu &quot;alvo Eventos&quot; para triagem contínua. </li><li> Adicionar populações para classificar no campo correspondente aos tubos (na janela &quot;Ordenar Layout&quot;). Populações lugar com freqüências mais elevadas no meio e aqueles com freqüências mais baixas nas extremidades. Defina as placas de tensão a 2.500 V. Durante a triagem, manter a coleta de amostra tUBEs no gelo. </li></ol> 4. celular Seleção <ol><li> Imediatamente antes da classificação, as células de filtro usando um filtro de 50 mm em uma previamente revestida 5 ml de polipropileno rodada tubo inferior. </li><li> Lavar o filtro extensivamente com 1-2 ml de tampão de triagem. </li><li> Células germinativas Ordenar fixas usando um classificador de células 488 nm equipado com laser seguindo o esquema gating detalhado na Figura 1. Recolha frações purificadas em 5 ml de polipropileno tubos de fundo redondo previamente revestidas no ponto 1 no gelo. <ol><li> Certifique-se de que 100-200 uL de SBF é mantido no interior dos tubos de recolha, para evitar a aderência das células escolhidas para a parede do tubo. </li><li> Certifique-se de que a concentração de saturação é SYTO16 para evitar flutuações de fluorescência durante o período de triagem. SYTO16 está saturando quando não há nenhuma outra variação de intensidade da coloração de células no Alexa Fluor 488 parâmetro (coloração de DNA) nos gráficos de triagem. Se necessário, adicione 1 ml deSYTO16 para o tubo de triagem para atingir a saturação. </li><li> Mostrar o total de eventos em um gráfico mostrando os seguintes parâmetros: número de eventos vs Alexa Fluor 488-A. </li><li> Seleccionar as células positivas de coloração de ADN com uma porta, como mostrado na Figura 1. <ol><li> Células Mostrar os porta 1 em um gráfico utilizando SSC-A vs FSC-A como parâmetros <ol><li> Selecione as células com Gate 2, como mostrado na Figura 1. </li><li> Células Mostrar os Gate 2 em um gráfico usando FSC-A vs Alexa Fluor 488-A como parâmetros. </li><li> Selecione as células com portão 5 e 6 no Portão Gate 2 gráfico, como mostrado na Figura 1. </li><li> Exibir separadamente Portão 5 e 6 no Portão gráficos usando Alexa Fluor 488-W vs Alexa Fluor 488-A como parâmetros. </li><li> Selecionar spermiogenesis passos 1-9 espermátides no Portão 5 gráfico e spermiogenesis passos 10-12 espermátides no portão 6 ilustrações, como mostrado na Figura 1. </li></ol></li><li> Displaycélulas do portão 1 em um gráfico usando FSC-A vs Alexa Fluor 488-A como parâmetros <ol><li> Selecione as células com Portão 3 e Gate 4, como mostrado na Figura 1. </li><li> Exibir separadamente Portão 3 e Gate 4 de gráficos usando Alexa Fluor 488-W vs Alexa Fluor 488-A como parâmetros. </li><li> Selecionar spermiogenesis passos 13-14 espermátides no gráfico Portão 3 e spermiogenesis passos 15-16 espermátides no gráfico Gate 4, como mostrado na Figura 1. </li></ol></li></ol></li></ol></li></ol>

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The authors declare no competing financial interests.

Células espermatogênicas foram sempre um desafio para o estudo, dada a complexidade do epitélio seminífero, bem como o sucesso limitado de cultura in vitro. Ao longo dos anos, muitas abordagens para purificar as células germinais de diferentes espécies foram desenvolvidos. Técnicas de sedimentação utilizando purificação gravidade com Percoll ou gradientes de albumina do soro bovino geralmente oferecem um bom rendimento de células germinais intactas, mas falta definição adequada entre alguns tipos ...

Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size. 
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.

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	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Isoflurane</td>
			<td style="width:119px;">ABBOT</td>
			<td style="width:129px;">05260-05</td>
			<td style="width:184px;">For mouse anesthesia before euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Fetal bovine serum</td>
			<td style="width:119px;">Wisent</td>
			<td style="width:129px;">90150</td>
			<td style="width:184px;">For tube coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">1X PBS</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">EDTA</td>
			<td style="width:119px;">BioShop</td>
			<td style="width:129px;">EDT</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">HEPES</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:129px;">H</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">100 % Ethanol</td>
			<td style="width:119px;">Les alcools de commerce</td>
			<td style="width:129px;">092-09-11N</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">SYTO 16</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:129px;">S7578</td>
			<td style="width:184px;">DNA staining</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">5 ml polypropylene round bottom tubes</td>
			<td style="width:119px;">BD Falcon</td>
			<td style="width:129px;">352063</td>
			<td style="width:184px;">Sorted cells collection</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">15 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">1500</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">50 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">5000</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">TEC4 anaesthetic vaporizer</td>
			<td style="width:119px;">Ohmeda</td>
			<td style="width:129px;">1160526</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">CO2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">C799117902</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">O2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">O254130501</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Homemade mouse gas chamber</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">40 &#181;m Falcon cell strainer</td>
			<td style="width:119px;">Corning Incorporated</td>
			<td style="width:129px;">352340</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">50-micron sample line filters</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">649049</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Vortex mixer</td>
			<td style="width:119px;">Labnet international, inc.</td>
			<td style="width:129px;">S0200</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dynac centrifuge</td>
			<td style="width:119px;">Clay Adams</td>
			<td style="width:129px;">101</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Celltrics 50 &#181;m filters</td>
			<td style="width:119px;">Partec</td>
			<td style="width:129px;">04-004-2327</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">488 nm laser-euipped cell sorter</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">FACSAria III</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:135px;">Accudop Fluorescent Beads</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">345249</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">Sorting Buffer: 1X PBS, 1mM EDTA pH 8.0, 25mM HEPES pH 7.0, 1%FBS</td>
			<td></td>
			<td></td>
			<td style="width:184px;">FBS is heat-inactivated. Make fresh solution, 0.22 &#956;m filtered and keep at 4&#176;C.</td>
		</tr>
	</tbody>
</table>

step-specific sorting of mouse spermatids by flow cytometry

A diferenciação dos espermatídeos rato é um processo crítico para a produção de uma gâmeta masculina funcional com um genoma intacto para ser transmitido à geração seguinte. Até agora, os estudos moleculares desta transição morfológica têm sido dificultados pela falta de um método que permite uma separação adequada dessas etapas importantes de diferenciação spermatid para análises subsequentes. Tentativas anteriores de gating adequada destas células usando citometria de fluxo pode ter sido difícil por causa de um aumento peculiar na fluorescência DNA em espermátides submetidos à remodelação da cromatina. Com base nesta observação, que fornecem detalhes de um simples fluxo de citometria de regime, permitindo a purificação reprodutível de quatro populações de espermatídeos rato fixas com etanol, cada um representando um estado diferente no processo de remodelação nuclear. Enriquecimento população é confirmado utilizando marcadores específicos passos e critérios morfológicos. Os espermatídeos purificados podem ser utilizados para genómico e proteomic analisa.

Gating estratégia utilizada com citometria de fluxo A Figura 1 representa a estratégia utilizada gating em citometria de fluxo para classificar quatro populações espermátide altamente puros. Resumidamente, as células com coloração positiva de ADN (Alexa Fluor 488-A) são primeiro seleccionadas com portão 1. Espermátides de spermiogenesis etapas 1-12 são seleccionado (porta 2) sobre um gráfico de po...

Watch this Scientific Journal Video about Step-specific Sorting of Mouse Spermatids by Flow Cytometry at JoVE.com

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Passo-específico Seleção de mouse Espermátides por citometria de fluxo

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

step-specific-sorting-of-mouse-spermatids-by-flow-cytometry

Research

JoVE Journal

Developmental Biology

10.4K visualizações.  Université de Sherbrooke. We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Vídeo: Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Identificação de progenitores eritromoelóides e sua progênie no embrião de mouse por citometria de fluxo

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Biologia do Desenvolvimento

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

Isolando e analisando células do pâncreas Mesênquima por citometria de fluxo

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Uma abordagem padronizada para a purificação multiespecífica de células germinais masculinas de mamíferos por dissociação mecânica do tecido e citometria de fluxo

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Uso de transplante de células-tronco hematopoiéticas para avaliar a origem da síndrome mielodisplásica

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Citometria de fluxo de viver-pilha em tempo real para investigar o influxo de cálcio, formação de poros e fagocitose pelos receptores P2X7 em células progenitoras neurais adulto

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

Isolação e mancha de keratinocytes da pele do rato para a análise específica do ciclo de pilha da expressão celular da proteína pela citometria maciça

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

Enriquecimento de espermatócitos de Pachytene e Spermatids de testes de camundongo usando equipamento de laboratório padrão

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Usando citometria de fluxo para detectar e quantificar a formação sanguínea alterada no zebrafish em desenvolvimento

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

FACS-isolamento e Cultura de Progenitores Fibro-Adipogênicos e Células-Tronco Musculares de Músculo Esquelético não perturbado e lesionado

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Identificação e Isolamento de Progenitores Eritróides da Unidade Formadora de Explosão e da Unidade Formadora de Colônias de Tecido de Camundongo por Citometria de Fluxo

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...