Soutenu par le Prix de développement de carrière à partir de la recherche pour prévenir la cécité, NIH EY 019 312, et subvention sans restriction de la recherche pour prévenir la cécité et EY 02687 (Département d&#39;ophtalmologie et de sciences visuelles à l&#39;Université Washington).

Faire électrodes <ol><li> Faire les électrodes d&#39;enregistrement à l&#39;aide d&#39;un tire-micropipette et polir les électrodes avec filament chauffant sous le microscope. Pour cône de la souris d&#39;aspiration d&#39;enregistrement unicellulaire, le diamètre intérieur de la pointe de l&#39;électrode est d&#39;environ um 6-7. </li><li> Préparer des électrodes de référence. Peser agar à 1% dans de l&#39;eau distillée. Faire fondre la solution d&#39;agar dans le bain d&#39;eau chaude. Remplissez les pipettes en verre (L = 100 mm, OD / ID = 1/0.75 mm) avec de l&#39;agar à 1% en utilisant une seringue. Solidifier la gélose à la température ambiante pendant 10 minutes. </li><li> Couper les pipettes en deux moitiés à l&#39;aide d&#39;un couteau de diamant. </li><li> Faire tremper les électrodes de référence en solution de référence pour au moins 24 heures à 4 ° C. </li></ol> Mise en place de l&#39;expérience <ol start="5"><li> Sombre adapter une souris dans une cage noire nuit. </li><li> Préparer 100 ml solution de référence et d&#39;une solution de perfusion L (voir ci-dessous) et filtrer la solution. </li><li> Dissoudre 0,1% de BSA et 10 mM de glucose dans 20 ml solution de référence. </li><li> Calibrer l&#39;intensité de la lumière du stimulateur optique en utilisant photomètre. </li><li> Montez la chambre d&#39;enregistrement sur la platine du microscope inversé. </li><li> Bubble la solution de perfusion avec 95% d&#39;O 2 / 5% CO 2, il incubation à 40 ° C bain d&#39;eau pour augmenter la solubilité du CO 2. La solution coule de la bouteille à la chambre d&#39;enregistrement en passant par une résistance en céramique, il le réchauffement à 36-38 ° C. Le chauffe-eau doit être située aussi près de la chambre d&#39;enregistrement que possible, idéalement au stade du microscope. </li><li> Ajustez le régulateur de débit pour un débit de environ 1-1,5 ml / min. </li><li> Remplir l&#39;électrode d&#39;enregistrement avec une solution de référence. Insérer l&#39;électrode dans un porte-électrode. Assurez-vous qu&#39;il n&#39;ya pas de bulles dans l&#39;électrode. Monter le support sur le headstage de l&#39;amplificateur. Connectez le titulaire d&#39;un dispositif d&#39;aspiration, qui est construit par le raccordement de tuyaux partiellement remplis d&#39;huile minérale à l&#39;orifice latéral du porte-électrodes. L&#39;autre extrémité du tuyau est relié à un petit réservoir d&#39;huile minérale, pour laquelle la pression peut être appliquée par la bouche. </li><li> Connectez l&#39;électrode de référence à l&#39;autre pôle de l&#39;headstage. Lier le couple thermique (TC) à l&#39;électrode de référence. Assurez-vous que les extrémités des électrodes et TC sont rapprochés pour que la lecture de température est considéré comme proche de la cellule que possible. </li><li> Allumez l&#39;éclairage infrarouge pour visualiser l&#39;électrode. L&#39;électrode est considérée par caméra infrarouge relié à un moniteur. Centre de l&#39;image d&#39;électrodes sur le champ visuel et de l&#39;écran en utilisant un micro-manipulateur. </li><li> Ajustez la position de l&#39;étroite pointe de l&#39;électrode de référence (2-4 mm) à l&#39;extrémité de l&#39;électrode d&#39;enregistrement. </li><li> Régler la tension DC pour le chauffage de telle sorte que la température de la solution est d&#39;environ 36-38 ° C. </li><li> Exécuter un test d&#39;étanchéité pour vérifier la résistance des électrodes d&#39;enregistrement, normalement environ 900 kQ. </li></ol> Isoler la rétine de souris <ol start="18"><li> Sous la lumière rouge sombre, euthanasier la souris adaptés à l&#39;obscurité avec le CO 2 et de dislocation cervicale. Marquer les globes oculaires à l&#39;aide de la pointe d&#39;un chalumeau de métal chaud en cautérisant le point le plus dorsal de la sclérotique légèrement. Énucléer les globes oculaires à l&#39;aide des ciseaux. Toutes les procédures subséquentes sont effectuées sous la lumière infrarouge. </li><li> Hemisect les globes oculaires sous microscope avec éclairage infrarouge et convertisseurs d&#39;images. </li><li> Retirez la cornée et le cristallin. Retirer autant du vitré que possible. </li><li> Pour enregistrer M-cône de réponses, enlever la partie ventrale de l&#39;œilleton 4 avec une lame de rasoir en juger par la marque de cautérisation. </li><li> Peler la rétine de la couche de l&#39;épithélium pigmentaire. </li><li> Aplatir la rétine et le placer sur le fond de la boîte de Pétri remplie avec 1-2 ml solution de référence. </li><li> Trancher la rétine en petits morceaux minces à l&#39;aide d&#39;une lame de rasoir et incuber la préparation dans une boîte étanche à la lumière saturée en oxygène pur. </li></ol> Enregistrements <ol start="25"><li> Éteignez le chauffe temporairement. Mettre le flux de la perfusion à un tuyau de dérivation pour empêcher la chambre de séchage. </li><li> Égoutter la plupart de la solution dans la cuve à l&#39;aide d&#39;un morceau de tissus Kimwipe. Transférer la suspension rétine à la chambre d&#39;enregistrement à l&#39;aide d&#39;une pipette en verre. </li><li> Attendez environ 2 minutes jusqu&#39;à ce que les tranches de la rétine s&#39;installer. Mettez la perfusion de retour sur. Allumez le chauffage. </li><li> Allumez l&#39;éclairage infrarouge pour visualiser la préparation. Recherche d&#39;un morceau de la rétine couché sur la chambre avec des segments externes des photorécepteurs qui sort (voir la figure par exemple). Dessine des segments intérieurs doucement dans l&#39;électrode par aspiration. Dans cette configuration, plusieurs noyaux et les segments intérieurs devrait être établi dans l&#39;électrode. Enregistrez le photoréponse d&#39;un flash lumineux pour tester si un segment de cône intérieur est dans l&#39;électrode et si la cellule est guérissenttes, à en juger par la cinétique de réaction et d&#39;amplitude. </li><li> Abaissez le réservoir de minéraux pour exercer une légère pression négative à la pointe de l&#39;électrode pour aider à maintenir le segment intérieur de l&#39;électrode. </li><li> Enregistrement test-éclair à partir des réponses aux DIM l&#39;intensité lumineuse une fois qu&#39;une cellule est jugée satisfaisante. Test clignote sont fournis à partir d&#39;un stimulateur calibré optique 5. L&#39;intensité du flash et la longueur d&#39;onde sont contrôlés par un ensemble de filtres de densité neutre et étroite bande de filtres d&#39;interférence, respectivement. Test de durée du flash (20 ms) est contrôlé par ordinateur axée sur les volets. </li></ol> Solutions <ol><li> Solution de perfusion Souris: 112,5 mM de NaCl, 3,6 mM de KCl, 2,4 mM de MgCl2, 1,2 mM de CaCl2, 10 mM HEPES (pH 7,4), 20 mM NaHCO 3, 3 mM Na succinate, 0,5 mM Na glutamate, 0,02 mM EDTA, et 10 mM de glucose. </li><li> Solution de l&#39;électrode d&#39;enregistrement de la souris: NaCl 140 mM, 3,6 mM de KCl, 2,4 mM de MgCl2, 1,2 mM CaCl 2, 3 mM HEPES (pH 7,4), 0,02 mM d&#39;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>

L&#39;enregistrement d&#39;aspiration mono-cellulaire de cellules photoréceptrices a été développé il ya 3 décennies. Il nous permet d&#39;enregistrer le changement trans-membranaire courant induit par une stimulation lumineuse sans pénétrer la membrane cellulaire. En raison de haute adhérence cellule-cellule, il est difficile d&#39;isoler saine seule tige et le cône de la rétine de souris, comme la rétine des amphibiens et il est difficile de trouver des cônes individuels en raison de la faible pourcentage...

single-cell suction recordings from mouse cone photoreceptors

Photorécepteurs Rod et cônes dans la rétine sont responsables de la détection de lumière. Dans l&#39;obscurité, des nucléotides cycliques-dépendants (CNG) des canaux dans le segment externe sont ouvertes et permettent aux flux de cations régulièrement vers l&#39;intérieur à travers la membrane, dépolariser la cellule. Exposition à la lumière déclenche la fermeture des canaux CNG, bloque le flux cation courant entrant, et donc les résultats de l&#39;hyperpolarisation cellulaire. Basé sur la polarité des photorécepteurs, une méthode d&#39;enregistrement d&#39;aspiration a été développé en 1970 que, contrairement à la classique technique de patch-clamp, ne nécessite pas de pénétrer la membrane plasmique<sup&gt; 1</sup&gt;. Dessin du segment externe dans une pipette en verre bien ajustés remplis de solution extracellulaire permet d&#39;enregistrer les changements en cours dans des cellules individuelles lors de test-éclair d&#39;exposition. Cependant, ce bien établi &quot;extérieur-segment-in (OS-in)&quot; enregistrement d&#39;aspiration n&#39;est pas adapté pour les enregistrements de cône de la souris, en raison de la faible pourcentage de cônes dans la rétine de souris (3%) et les difficultés à identifier le cône segments externes. Récemment, un segment interne-dans (IS-in) d&#39;enregistrement de configuration a été développé pour tracer le segment intérieur / région nucléaire du photorécepteur dans la pipette d&#39;enregistrement<sup&gt; 2,3</sup&gt;. Dans cette vidéo, nous allons montrer comment enregistrer à partir individuels photoresponses cône de souris à l&#39;aide d&#39;électrodes à aspiration simple cellule.

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

Nous allons montrer comment enregistrer les réponses flash à partir de cônes de souris en utilisant une électrode de succion.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

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

11.7K vues.  Washington University in St. Louis, School of Medicine. Nous allons montrer comment enregistrer les réponses flash à partir de cônes de souris en utilisant une électrode de succion.

Vidéo: 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.

Méthodes pour les enregistrements patch clamp Capacité du calice

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

Recherche

Biologie

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.

Préparation des suspensions unicellulaires à partir de rate de souris avec le dissociateur 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.

Préparation standardisée des suspensions unicellulaires provenant de tissus pulmonaires de la souris en utilisant les dissociateur 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.

Génération d&#39;une seule cellule suspensions de tissus de souris de neurones

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.

Profilage Single Cell transcriptionnelle des cardiomyocytes de souris adultes

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

TRANS-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

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.

Isolement de cellules endothéliales de la lumière des artères carotides de souris pour des expériences multi-omiques unicellulaires

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.

Préparation de suspension unicellulaire de l’intestin de tilapia du Nil pour séquençage unicellulaire

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.

Isolement et analyse transcriptome de types de cellules végétales

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