失明とEY 02687（ワシントン大学眼科視覚科学の部）防止のための研究から失明、NIHの助成金EY 019312、および無制限の助成金を防止するための研究からキャリア開発賞でサポートされています。

電極を作る<ol><li>マイクロピペットプラーを使用して、記録電極を作成し、顕微鏡下で加熱フィラメントと電極を磨く。マウスの錐体単一細胞吸引録音の場合、電極先端の内径は6-7μm程度である。 </li><li>参照電極を準備する。蒸留水で1％の寒天を量る。湯浴の寒天液を溶かす。シリンジを用いて1％の寒天をガラスピペットを（L = 100 mmの外径/ ID = 1/0.75 mm）を記入します。 10分間室温で寒天を固める。 </li><li>ダイヤモンドナイフを使用して半分にピペットをカット。 </li><li> 4℃で少なくとも24時間は、基準溶液中での参照電極を浸し℃に</li></ol>実験のセットアップ<ol start="5"><li>暗い晩黒ケージでマウスを適応させる。 </li><li> 100 mLの標準液と1リットルの灌流液を（下記参照）を準備し、ソリューションをフィルタリングする。 </li><li> 0.1％BSA、20 mLの標準液中の10mMグルコースを溶解する。 </li><li>光度計を用いた光刺激装置の光強度を校正します。 </li><li>倒立顕微鏡のステージ上で録音室をマウントします。 </li><li> 95％O 2 / 5％CO 2のバブル灌流液は、CO 2の溶解度を高めるために40 ° Cの水浴中でインキュベートする。解決策は、ボトル36から38に加温し、セラミック抵抗を通過する記録チャンバー℃に流れるヒーターは、理想的に顕微鏡の段階で、可能な限りの記録室の近くに配置する必要があります。 </li><li> 1〜1.5程度mL /分の流量のフローレギュレータを調整します。 </li><li>参照溶液と記録電極を埋める。電極ホルダに電極を挿入します。電極内に気泡がないことを確認します。アンプのヘッドステージ上にホルダーをマウントします。部分的に電極ホルダーのサイドポートにミネラルオイルを充填したチューブを接続することによって構築されている吸引装置、にホルダーを接続してください。チューブのもう一方の端は口の中でどの圧力を適用することができるため、鉱物油の小さなタンクに接続されている。 </li><li>ヘッドステージの他の極に参照電極を接続してください。参照電極に熱カップル（TC）をバインドします。電極とTCのヒントが接近して温度の読み取りが可能な限り細胞に近いとみなされるようになっていることを確認します。 </li><li>電極を視覚化する赤外線照明の電源を入れます。電極は、モニターに接続されている赤外線カメラによって表示されます。視野上で、マイクロマニピュレータを用いてモニターの中心電極画像。 </li><li>記録電極の先端への参照電極の先端に近い（2-4 mm）の位置を調整します。 </li><li>溶液の温度が36から38℃程度になるようにヒータのDC電圧を調整する</li><li>通常、900kΩの約、記録電極の抵抗をチェックするためにシールのテストを実行します。 </li></ol>マウスの網膜を分離<ol start="18"><li>暗赤色灯の下で、CO 2と頚椎脱臼で暗順応マウスを安楽死させる。やや強膜の最も背点を焼灼による溶銑のトーチの先端を使って眼球をマーク。はさみを使って眼球を摘出。後続のすべての手順は、赤外光の下で実行されます。 </li><li>赤外線照明と画像コンバータと顕微鏡下での眼球を二等分する。 </li><li>角膜とレンズを取り外します。できるだけ硝子体をできるだけ多く削除します。 </li><li> M -錐体の応答を記録するには、焼灼マークから判断してかみそりの刃でアイカップ4の腹側部分を削除してください。 </li><li>色素上皮層から網膜ピール。 </li><li>網膜を平らに1〜2 mLの標準液で満たされたペトリ皿の底の上に置きます。 </li><li>カミソリの刃を使用して、小さな薄い部分に網膜をスライスし、純酸素で飽和遮光ボックスの準備をインキュベートする。 </li></ol>レコーディング<ol start="25"><li>一時的にヒーターの電源を切ります。乾燥室を防ぐためにバイパス管に血流の流れを切り替える。 </li><li>キムワイプ組織片を用いてチャンバー内の溶液のほとんどを排出します。ガラスピペットを使用して録音室に網膜の懸濁液を移す。 </li><li>網膜のスライスが落ち着くまで、約2分間待ちます。背面の血流を切り替えます。ヒーターの電源を入れます。 </li><li>準備を視覚化する赤外線照明の電源を入れます。突き出光受容体の外側のセグメントでチャンバーの上に横たわっ網膜の部分のための検索（例の図を参照）。吸引によって電極に軽く内側のセグメントを描画します。この構成では、いくつかの核と内側のセグメントは、電極に描画する必要があります。円錐内部セグメントが電極内にあるかどうかと、セルが治癒されているかどうかをテストするために明るいフラッシュに光応答を記録汝、応答速度と振幅によって判断する。 </li><li>電極の内側のセグメントを保持するための電極の先端にわずかな負圧を適用するには、ミネラルの貯水池を下ろします。 </li><li>薄暗いから明るい強度への記録テストフラッシュ応答良好な細胞が発見されれば。テストの点滅は、校正済み光刺激装置5から提供されています。フラッシュの強度と波長はそれぞれ、NDフィルターと狭帯域の干渉フィルターのセットによって制御されています。テスト発光持続時間（20ミリ）は、コンピュータ主導のシャッターで制御されます。 </li></ol>ソリューション<ol><li>マウスの灌流液：112.5 mMのNaCl、3.6 mMの塩化カリウム、2.4 mMのMgCl 2、1.2 mMのCaCl 2を 、10mMのHEPES（pH7.4）を、20mMのNaHCO 3を 、3 mMのコハク酸ナトリウム、0.5mMのナトリウムグルタミン酸、0.02mMのEDTA、および10mMのグルコース。 </li><li>マウスの記録電極液：140 mMのNaCl、3.6 mMの塩化カリウム、2.4 mMのMgCl 2、1.2 mMのCaCl 2を 、3 mMのHEPES（pH7.4）に、0.02mMの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>

視細胞から単一細胞吸引の記録は3年前に開発されました。それは細胞膜を貫通することなく、光刺激により誘発される膜貫通電流変化を記録することを可能にします。が高いために細胞間接着のために、それは両生類の網膜のようなマウスの網膜から健康なシングルロッドとコーンを分離することは困難であり、それは低い割合（3％）と小さいサイズのため、個々の錐体を見つけるのは難?...

single-cell suction recordings from mouse cone photoreceptors

網膜の桿体と錐体光受容体は、光検出のための責任があります。暗闇の中で、外側のセグメントにおける環状ヌクレオチド依存性（CNG）チャネルが開いていて、陽イオンが細胞を脱分極、膜を横切って内側に着実に流れるようになります。光露光では、CNGチャネル、ブロック内向きの陽イオン電流の流れ、および従って細胞過分極の結果の閉鎖をトリガします。光受容体の極性に基づいて、吸引の記録方式は、古典的パッチクランプ法とは異なり、原形質膜を貫通する必要がない、1970年代に開発された<sup&gt; 1</sup&gt;。細胞外液を満たしたしっかりフィットガラスピペットに外側のセグメントを描画するテスト - ストロボ調光時に個々の細胞の電流変化を記録することができます。しかし、この十分に確立された&quot;外側のセグメントイン（OS - IN）&quot;吸引録音ため、マウスの網膜の錐体の割合が低い（3％）とコーンの同定に関する問題点から、マウスのコーンの録音には適していません外側のセグメント。最近、内側セグメントの（IS -の）記録の設定は、録音のピペットに内側のセグメント/光受容体の核領域を描画するために開発された<sup&gt; 2,3</sup&gt;。このビデオでは、我々は単一細胞吸引電極を用いて個々のマウスのコーンphotoresponsesから録音する方法を示します。

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

我々は、吸引電極を用いてマウスのシングルコーンからフラッシュの応答を記録する方法を説明します。

マウスの錐体光受容体からの単一細胞サクション録音

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 ビュー.  Washington University in St. Louis, School of Medicine. 我々は、吸引電極を用いてマウスのシングルコーンからフラッシュの応答を記録する方法を説明します。

ビデオ: 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.

がくからパッチクランプの静電容量の録音のための方法

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

生物学

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.

アダルトショウジョウバエの脳から全細胞レコーディング

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.

gentleMACS Dissociatorとマウス脾臓からの単細胞懸濁液の調製

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.

gentleMACS Dissociatorを使用してマウスの肺組織からの単細胞懸濁液の標準化の準備

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.

マウス神経組織から単細胞懸濁液の生成

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.

成体マウスの心筋細胞の単一細胞転写プロファイリング

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.

人工多能性幹細胞に由来するマウスの作製

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.

シングルセルマルチオミクス実験のためのマウス頸動脈内腔からの内皮細胞の単離

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.

シングルセルシーケンシングのためのナイルティラピア腸からのシングルセル懸濁液調製(英語)

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.

植物細胞タイプの単離とトランスクリプトーム解析

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