실명과 어이 02687을 (워싱턴 대학에서 안과 &amp; 비주얼 과학 계열) 방지하기 위해 연구에서 실명, NIH 그랜트 어이 019,312, 그리고 무제한 부여를 방지하기 위해 연구에서 경력 개발 상을 지원.

전극 만들기 <ol><li> micropipette의 풀러를 사용하여 기록 전극을 만들어 현미경으로 가열 필라멘트와 전극을 폴란드어. 마우스 콘 단일 셀 흡입 녹음, 전극 팁의 내부 직경 6-7 μm의 약입니다. </li><li> 참조 전극을 준비합니다. 증류수 1 %의 한천을 나가는거야. 온수 욕조에있는 한천 솔루션을 용융. 주사기를 사용하여 1 %의 한천과 유리 pipettes을 (L = 100mm, OD / ID = 1/0.75 ㎜) 입력합니다. 10 분 상온에서 한천을 응고. </li><li> 다이아몬드 나이프를 사용하여 반쪽에 pipettes 컷. </li><li> 4 최소 24 시간에 대한 참조 솔루션에서 참조 전극을 소크 ° C. </li></ol> 실험 설정 <ol start="5"><li> 다크 하룻밤 검은 새장에 마우스를 적응. </li><li> 100 ML 참조 솔루션 1 L 재관류 솔루션 (아래 참조) 준비와 솔루션을 필터링합니다. </li><li> 0.1 % BSA 20 ML 참조 솔루션 10 MM의 혈당을 디졸브. </li><li> 광도계를 사용하여 광학 자극기의 빛을 강도를 보정합니다. </li><li> 거꾸로 현미경의 무대에서 기록 챔버를 탑재합니다. </li><li> 95% O 2 / 5% CO 2와 버블 재관류 솔루션은 CO 2의 용해도를 증가 40 ° C 물 목욕에 품어. 이 솔루션은 병 36-38로 온난 세라믹 저항을 통과 기록 챔버 ° C.에 흐르는 히터가 이상적으로 현미경의 무대에서 가능한 녹화 실로 가까운 위치해야합니다. </li><li> 1-1.5에 대해 ML / 분 유량에 대한 유량 조절기를 조정합니다. </li><li> 참조 솔루션으로 기록 전극을 입력합니다. 전극 홀더에 전극을 삽입합니다. 전극에 거품이 없습니다 있는지 확인합니다. 앰프의 headstage에 홀더를 탑재합니다. 부분적으로 전극 홀더의 사이드 포트에 미네랄 오일로 채워진 튜브를 연결하여 내장된 흡입 장치에 홀더를 연결합니다. 관의 다른 쪽 끝을는 입으로하는 압력을 적용할 수있는, 미네랄 오일의 작은 저수지에 연결되어 있습니다. </li><li> headstage의 다른 극에 대한 참조 전극을 연결합니다. 참조 전극을 열 부부 (TC)를 바인딩합니다. 전극과 TC 팁 가까이 함께 온도 읽기가 가능한 세포에 가까운 촬영되도록 있는지 확인합니다. </li><li> 전극을 시각화하기 위해 적외선 조명을 켭니다. 전극은 모니터에 연결된 적외선 카메라로 볼 수 있습니다. 시야와 마이크로 조작하는 사람을 사용하여 모니터의 중심 전극 이미지. </li><li> 기록 전극 팁을 참조 전극 팁 클로즈 (2~4밀리미터)의 위치를​​ 조정합니다. </li><li> 솔루션의 온도가 36-38에 대한 ° C.되도록 히터에 대한 DC 전압을 조정 </li><li> 일반적으로 900 kΩ에 대한, 기록 전극의 저항을 확인하는 인감 테스트를 실행합니다. </li></ol> 마우스 망막을 분리 <ol start="18"><li> 희미한 붉은 불빛 아래, CO 2 경추 전위와 어두운 적응 마우스를 안락사. 약간 공막엔의 가장 지느러미 지점을 cauterizing하여 뜨거운 금속 토치의 팁을 사용하여 시선을 표시합니다. 가위를 사용하여 눈알을 명백히하다. 이후의 모든 절차는 적외선에 따라 수행됩니다. </li><li> 적외선 조명 및 이미지 컨버터와 함께 현미경으로 눈알을 Hemisect. </li><li> 각막과 렌즈를 제거합니다. 가능한 유리의 많은를 제거합니다. </li><li> M - 원뿔 응답을 기록하려면, 소작 마르크에서 심사 면도날로 세안 컵 4 복부 부분을 제거합니다. </li><li> 안료 상피 층에서 망막 껍질. </li><li> 망막을 평평하게하고 1-2 ML 참조 솔루션 가득 페트리 접시의 바닥에 놓으십시오. </li><li> 면도날을 사용하여 작은 얇은 조각으로 망막를 슬라이스하고 순수한 산소와 포화 빛을 꽉 상자에서 준비를 품어. </li></ol> 녹음 <ol start="25"><li> 일시적으로 히터의 전원을 끕니다. 건조에서 약실을 방지하기 위해 우회 튜브에 재관류 흐름을 전환합니다. </li><li> Kimwipe 조직의 조각을 사용하여 챔버의 솔루션 대부분의 드레인. 유리 피펫을 사용하여 녹음 챔버에 망막의 현탁액을 전송합니다. </li><li> 망막 슬라이스가 정착 때까지 약 2 분 동안 기다리십시오. 다시 재관류를 전환합니다. 히터를 켭니다. </li><li> 준비를 시각화하는 적외선 조명을 켜십시오. photoreceptor 외부 세그먼트 (예 : 그림 참조) 붙어서 챔버에 누워 망막의 일부를 검색합니다. 흡입에 의해 전극에 부드럽게 내부 세그먼트를 그립니다. 이 구성에서 여러 개의 핵 및 내부 세그먼트는 전극에 그려해야합니다. 콘 내부 세그먼트는 세포 치료 여부를 전극에 있으며 있는지 여부를 테스트하려면 밝은 플래시로 photoresponse를 기록네, 반응 속도론 및 진폭에 의해 판단. </li><li> 전극의 내부 세그먼트를 개최 도움이되는 전극의 끝부분에 약간의 부정적인 압력을 적용하기 위해 광물 저수지를 낮춥니다. </li><li> 기록 테스트 - 플래시 응답 밝은 강도에 희미에서 좋은 휴대폰이 발견되면. 테스트 섬광은 보정 광학 자극기 5 제공됩니다. 플래시 강도와 파장은 각각 중립 밀도 필터와 좁은 밴드 간섭 필터의 집합에 의해 제어됩니다. 시험 기간은 플래시 (20 MS) 컴퓨터 기반의 셔터에 의해 제어됩니다. </li></ol> 솔루션 <ol><li> 마우스 재관류 솔루션 : 112.5 MM NaCl, 3.6 MM KCl, 2.4 MM MgCl 2, 1.2 MM CaCl 2, 10 MM HEPES (산도 7.4), 20 MM NaHCO 3, MM succinate 나, 0.5 MM 나 조미료, 0.02 MM EDTA (에틸렌 다이아 민 테트라 초산), 그리고 10 MM 포도당. </li><li> 마우스 기록 전극 솔루션 : 140 MM NaCl, 3.6 MM KCl, 2.4 MM MgCl 2, 1.2 MM CaCl 2, 3 MM HEPES (산도 7.4), 0.02 MM EDTA (에틸렌 다이아 민 테트라 초산). </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>

photoreceptor 세포에서 단일 세포 흡입 기록은 3 년 전 개발되었습니다. 이것은 세포막을 관통하지 않고 빛의 자극에 의​​해 유도된 횡단 막 전류 변화를 기록하는 데 있습니다. 때문에 높은 세포 - 세포 부착, 이건 수륙 양용 망막과 같은 마우스 망막에서 건강한 하나의 막대와 원뿔을 분리하기 어려운이며 그것은 낮은 비율 (3 %)과 작은 크기로 인해 각각의 원뿔을 찾을 수 어렵습니다. 내부 세그먼...

single-cell suction recordings from mouse cone photoreceptors

망막에있는 막대와 원뿔 photoreceptors 빛을 감지 책임이 있습니다. 어둠에서 바깥쪽 구간에 주기적 염기 - 게이 티드 (CNG) 채널 오픈하고 양이온이 세포 depolarizing, 멤브레인 안쪽에 걸쳐 꾸준히 흐름을 수 있습니다. 라이트 노출은 CNG 채널, 블록 안쪽 양이온 전류 흐름, 따라서 세포 hyperpolarization 결과의 폐쇄를 실행시킵니다. photoreceptors의 극성에 따라 흡입 녹음 방법은 고전적인 패치 - 클램프 기법과는 달리, 플라즈마 막을 관통 필요하지 않습니다, 1970 년대에 개발되었습니다<sup&gt; 1</sup&gt;. 세포외 솔루션으로 가득한 단단히 맞는 유리 피펫에 외부 세그먼트를 그리기 테스트 - 플래시 노출에 따라 각각의 세포에서 현재 변경 내용을 녹음하실 수 있습니다. 그러나,이 잘 구축 &quot;객관적인 세그먼트 인 (OS 인)&quot;흡입 녹음 때문에 마우스 망막의 원뿔의 낮은 비율 (3 %)과 원추형을 식별의 어려움, 마우스 콘 레코딩에 적합하지 않습니다 외부 세그먼트. 최근 내부 세그먼트 기능 (IS -)를 녹화 설정은 녹화 피펫으로 내부 세그먼트 / photoreceptor의 핵 영역을 그리기 위해 개발된<sup&gt; 2,3</sup&gt;. 이 비디오에서는, 우리는 단일 셀 흡입 전극을 사용하여 개별 마우스 콘 photoresponses에서 기록하는 방법을 보여줍니다.

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

우리는 흡입 전극을 사용하여 단일 마우스 콘에서 플래시 응답을 기록하는 방법을 보여줍니다.

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

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 조회수.  Washington University in St. Louis, School of Medicine. 우리는 흡입 전극을 사용하여 단일 마우스 콘에서 플래시 응답을 기록하는 방법을 보여줍니다.

비디오: 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.

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

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

연구

생물학

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 Dissociator과 마우스 비장에서 단일 셀 Suspensions의 준비

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 Dissociator를 사용하여 마우스 폐 조직에서 단일 셀 Suspensions의 표준화 준비

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.

마우스 신경 조직에서 단일 셀 Suspensions의 생성

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.

성인 마우스 Cardiomyocytes의 단세포 Transcriptional 프로파일

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.

유도 만능의 줄기 세포에서 파생 마우스의 생성

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.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

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.

단일 세포 다중 오믹스 실험을 위한 마우스 경동맥 내강에서 내피 세포 분리

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.

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.

식물 세포 유형의 분리 및 전사체 분석

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

Methods Article

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩