Suportado pelo Prémio Carreira de Desenvolvimento de Pesquisa para prevenir a cegueira, NIH conceder EY 019.312, e concessão irrestrita de Pesquisa para prevenir a cegueira e EY 02.687 (Departamento de Oftalmologia &amp; Visual Sciences da Universidade de Washington).

Fazendo eletrodos <ol><li> Fazer a gravação usando eletrodos de um puxador de micropipeta e polonês os eletrodos com filamento de aquecimento sob o microscópio. Para mouse gravação de sucção cone de uma única célula, o diâmetro interno da ponta do eletrodo é de cerca de M 6-7. </li><li> Prepare eletrodos de referência. Pesar agar 1% em água destilada. Derreta a solução ágar-ágar em banho de água quente. Preencha as pipetas de vidro (L = 100 mm, OD / ID = 1/0.75 mm) com 1% agar usando seringa. Solidificar o ágar em temperatura ambiente por 10 minutos. </li><li> Cortar as pipetas em metades usando uma faca de diamante. </li><li> Mergulhe os eletrodos de referência na solução de referência para pelo menos 24 horas a 4 ° C. </li></ol> Configurando o experimento <ol start="5"><li> Escuro adaptar um rato em uma gaiola preta durante a noite. </li><li> Preparar 100 mL de solução de referência e uma solução de perfusão L (veja abaixo) e filtrar a solução. </li><li> Dissolver 0,1% BSA e 10 mM de glicose em 20 mL de solução de referência. </li><li> Calibrar a intensidade de luz do estimulador óptico utilizando fotômetro. </li><li> Monte a câmara de gravação no palco do microscópio invertido. </li><li> Solução da bolha da perfusão com 95% O 2 / 5% CO 2, incubar em banho-maria 40 ° C para aumentar a solubilidade de CO 2. A solução flui da garrafa para a câmara de gravação passando por um resistor de cerâmica aquecendo-a 36-38 ° C. O aquecedor deve estar localizado o mais próximo da câmara de gravação possível, de preferência na fase do microscópio. </li><li> Ajuste o regulador de fluxo de um fluxo de cerca de 1-1,5 mL / min. </li><li> Preencher o eletrodo de registro com a solução de referência. Insira o eletrodo em um porta-eletrodo. Certifique-se que não há bolhas no eletrodo. Montar o suporte no headstage do amplificador. Conecte o seu titular a um dispositivo de sucção, que é construído ligando tubo parcialmente cheio com óleo mineral ao lado da porta do porta-eletrodo. A outra extremidade do tubo é conectado a um pequeno reservatório de óleo mineral, para que a pressão pode ser aplicada por via oral. </li><li> Conecte o eletrodo de referência para o outro pólo da headstage. Vincular o casal térmica (TC) para o eletrodo de referência. Certifique-se que as pontas de eletrodos e TC estão juntos para que a leitura da temperatura é tida como próxima da célula como possível. </li><li> Ligue a iluminação por infravermelhos para visualizar o eletrodo. O eletrodo é visto pela câmera de infravermelho conectado a um monitor. Centro da imagem o eletrodo no campo visual e do monitor usando um micro-manipulador. </li><li> Ajustar a posição do próximo da ponta do eletrodo de referência (2-4 mm) para a ponta do eletrodo de gravação. </li><li> Ajustar a tensão DC para o aquecedor de modo que a temperatura da solução é de cerca de 36-38 ° C. </li><li> Executar um teste de vedação para verificar a resistência do eletrodo de registro, normalmente cerca de 900 kW. </li></ol> Isolar a retina do rato <ol start="18"><li> Sob luz vermelha ofuscante, euthanize o mouse de adaptação ao escuro com CO 2 e deslocamento cervical. Marcar o globo ocular com a ponta de uma tocha de metal líquido, cauterização o ponto mais dorsal da esclera ligeiramente. Enuclear do globo ocular usando uma tesoura. Todos os procedimentos subseqüentes são realizadas sob a luz infravermelha. </li><li> Hemisect os globos oculares em microscópio com iluminação infravermelha e conversores de imagem. </li><li> Remova a córnea ea lente. Remover o máximo do vítreo possível. </li><li> Para gravar M cone-respostas, remova a parte ventral da ocular 4 com uma lâmina de barbear a julgar pela marca cautério. </li><li> Descasque a retina da camada de epitélio pigmentar. </li><li> Achatar a retina e colocá-lo na parte inferior da placa de Petri cheia de 1-2 mL solução de referência. </li><li> Fatia da retina em pequenos pedaços finos usando uma lâmina de barbear e incube a preparação em uma caixa à prova de luz saturada com oxigênio puro. </li></ol> Gravações <ol start="25"><li> Desligue o aquecedor temporariamente. Mudar o fluxo de perfusão a uma tubulação de desvio para evitar que a câmara de secagem. </li><li> Escorra a maioria da solução na câmara usando um pedaço de tecido Kimwipe. Transferir a suspensão retina para a câmara de gravação usando uma pipeta de vidro. </li><li> Aguarde cerca de 2 minutos até as fatias de retina sossegar. Mudar a perfusão de volta. Ligar o aquecedor. </li><li> Ligue iluminação por infravermelhos para visualizar a preparação. Busca de um pedaço de retina deitado na câmara com segmentos fotorreceptoras exterior saindo (ver figura exemplo). Desenhar segmentos interior suavemente o eléctrodo por sucção. Nesta configuração, vários núcleos e segmentos internos devem ser atraídos para o eletrodo. Registre a photoresponse a um flash brilhante para testar se um segmento de cone interior é no eletrodo e se a célula é curartua, a julgar pela cinética de resposta e amplitude. </li><li> Inferior do reservatório mineral de aplicar uma ligeira pressão negativa para a ponta do eletrodo para ajudar a segurar o segmento interno no eletrodo. </li><li> Registro de teste flash-respostas de dim com a intensidade luminosa, uma vez por celular bom é encontrado. Flashes de teste são fornecidos a partir de um estimulador calibrado óptica 5. Flash intensidade e comprimento de onda são controladas por um conjunto de filtros de densidade neutra e filtros de interferência de banda estreita, respectivamente. Teste de flash duração (20 ms) é controlado por computador-driven persianas. </li></ol> Soluções <ol><li> Solução de perfusão do mouse: 112,5 mM NaCl, 3,6 mM KCl, 2,4 mM MgCl 2, 1,2 mM CaCl 2, 10 mM HEPES (pH 7,4), 20 mM NaHCO 3, 3 mM Na succinato, 0,5 mM Na glutamato, 0,02 mM EDTA, e 10 mM de glicose. </li><li> Rato solução eletrodo de registro: 140 mM NaCl, 3,6 mM KCl, 2,4 mM MgCl 2, 1,2 mM CaCl 2, 3 mM HEPES (pH 7,4), 0,02 mM EDTA. </li></ol>

<ol>
	<li>Yau, K. W., Lamb, T. D., Baylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+fluctuations+in+membrane+current+of+single+toad+rod+outer+segments.">Light-induced fluctuations in membrane current of single toad rod outer segments.</a> Nature. 269, 78-80 (1977).</li><li>Nikonov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptors+of+Nrl+-/-+mice+coexpress+functional+S-+and+M-cone+opsins+having+distinct+inactivation+mechanisms.">Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.</a> J Gen Physiol. 125, 287-304 (2005).</li><li>Nikonov, S. S., Kholodenko, R., Lem, J., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+features+of+the+S-+and+M-cone+photoreceptors+of+wild-type+mice+from+single-cell+recordings.">Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.</a> J Gen Physiol. 127, 359-374 (2006).</li><li>Applebury, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+cone+photoreceptor:+a+single+cone+type+expresses+both+S+and+M+opsins+with+retinal+spatial+patterning.">The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.</a> Neuron. 27, 513-523 (2000).</li><li>Cornwall, M. C., Fein, A., MacNichol, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+that+underlie+bleaching+and+background+adaptation.">Cellular mechanisms that underlie bleaching and background adaptation.</a> J Gen Physiol. 96, 345-372 (1990).</li><li>Calvert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransduction+in+transgenic+mice+after+targeted+deletion+of+the+rod+transducin+alpha+-subunit.">Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.</a> Proc Natl Acad Sci U S A. 97, 13913-13918 (2000).</li></ol>

De uma única célula de gravação de sucção de células fotorreceptoras foi desenvolvido três décadas atrás. Ele nos permite gravar trans-membrana mudança corrente induzida pela estimulação luminosa sem penetrar a membrana celular. Por causa da adesão celular de alta, é difícil isolar vara única saudável e cone da retina do rato como retina de anfíbios e é difícil encontrar cones individuais devido ao baixo percentual (3%) e tamanho pequeno. O segmento interno--in (IS-in) método de gravação supera e...

single-cell suction recordings from mouse cone photoreceptors

Haste e cone fotorreceptores na retina são responsáveis ​​pela detecção de luz. Na escuridão, nucleotídeo cíclico-gated (CNG) no segmento de canais externos são abertos e permitem o fluxo constante cátions para o interior através da membrana, despolarizando a célula. Exposição à luz provoca o fechamento dos canais de CNG, bloqueia o fluxo para dentro cação atual, e, portanto, resulta em hiperpolarização celular. Com base na polaridade de fotorreceptores, um método de gravação de sucção foi desenvolvido em 1970 que, ao contrário da técnica patch-clamp clássico, não requer penetrar a membrana plasmática<sup&gt; 1</sup&gt;. Desenhar o segmento externo em uma pipeta de vidro bem presas preenchido com solução extracelular permite a gravação das mudanças atuais em células individuais por testes de flash-exposição. No entanto, este bem estabelecido &quot;outer-segmento-in (OS-in)&quot; de gravação de sucção não é adequado para gravações cone mouse, por causa da baixa porcentagem de cones na retina do rato (3%) e as dificuldades na identificação do cone segmentos externos. Recentemente, um segmento interno--in de configuração (IS-in) de gravação foi desenvolvido para desenhar o segmento de interior / região nuclear do fotoreceptor para a pipeta de gravação<sup&gt; 2,3</sup&gt;. Neste vídeo, vamos mostrar como gravar a partir photoresponses mouse individual cone usando uma única célula do eletrodo de sucção.

Watch this Scientific Journal Video about Single-cell Suction Recordings from Mouse Cone Photoreceptors at JoVE.com

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Vamos mostrar como gravar respostas flash de cones do mouse usando um eletrodo de sucção.

Gravações de uma única célula de sucção de cones fotorreceptores rato

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

single-cell-suction-recordings-from-mouse-cone-photoreceptors

Research

JoVE Journal

Biology

11.6K visualizações.  Washington University in St. Louis, School of Medicine. Vamos mostrar como gravar respostas flash de cones do mouse usando um eletrodo de sucção.

Vídeo: Single-cell Suction Recordings from Mouse Cone Photoreceptors

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Métodos para Recordings patch-clamp Capacitância da Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Biologia

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

Todo Recordings celular do cérebro de adultos Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Preparação de uma única célula Suspensões de Spleen rato com o Dissociator gentleMACS

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

Preparação padronizado de uma única célula Suspensões do tecido pulmonar do rato usando o Dissociator gentleMACS

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Geração de Single-Cell Tissue Suspensões de rato Neural

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Profiling única célula transcricional dos cardiomiócitos de rato adulto

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Isolamento de células endoteliais do lúmen de artérias carótidas de camundongos para experimentos multi-ômicos unicelulares

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Preparação de Suspensão de Célula Única do Intestino de Tilápia do Nilo para Sequenciamento de Célula Única

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

Isolamento e Análise do Transcriptoma de Tipos Celulares Vegetais

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...