무균 조건 하에서 해결하려면, 그것은 층류 후드의 모든 단계를 수행하는 것이 좋습니다. 1. 자료 <ul><li> 신경 조직 분리 키트 (P) (구성 : 솔루션 1,2, 3, 4, 스토리지 버퍼) </li><li> 사전 분리 필터 </li><li> (선택 사항) Myelin 제거 비즈 </li><li> 베타 - 메르 캅 토 에탄올 </li><li> gentleMACS Dissociator </li><li> C 튜브 </li><li> MACSmix 튜브의 회전 </li><li> HBSS (W / O) </li><li> HBSS (W) </li></ul><ol><li> 이전 프로토콜을 시작하려면 다음 솔루션을 준비 : <ol><li> 이가의 양이온없이 행크스 &#39;버퍼 소금 솔루션 칼슘 2 + 또는 MG 2 + (HBSS (W / O) </li><li> HBSS, 표준, 즉 칼슘 2 +와 MG 2 + (HBSS (W) </li></ol></li><li> 이후 애플 리케이션에 대한 관심의 항원 에피토프에 따라 중 Papain 기반의 신경 조직 분리 키트 (P) 또는 트립신 기반의 신경 조직 분리 키트 (T)를 사용합니다. </li><li> 신경 조직 분리 키트에서 다음 솔루션을 준비 : <ol><li> 해결 방법 2 : 0.067 mm까지 베타 메르 캅 토 에탄올을 추가 </li><li> 솔루션 4 : 0.7 ML 저장소 버퍼 (키트에서 제공)에 분말을 용해, 부드럽게 (소용돌이하지 않음) 믹스 </li><li> 효소 배합 1 : C 튜브와 10-15 분 부화에 피펫 1.9 ML 솔루션이 50 μl 솔루션 1 (키트에서 모두) 37 ° C. 이것은 뇌 조직을 떼어 놓다 400 밀리그램하기에 충분합니다. </li></ol></li></ol> 2. 신경 조직을 Dissociating <ol><li> 1 ML HBSS (W / O)이있는 튜브의 뇌 조직을 달다 </li><li> preheated 효소 혼합 1 1,950 μL를 포함하는 C 튜브에 마우스 머리를 전송합니다. (참고 :이 프로토콜은 ≤ 400 밀리그램에 대한 기록이지만, 솔루션 볼륨 C 튜브마다 뇌 조직의 1600 MG 최대 수용 확장할 수 있습니다) </li><li> gentleMACS의 Dissociator에 C 튜브를 삽입하고 프로그램 &quot;m_brain_01&quot;를 실행합니다. </li><li> 37 15 분에 대한 회전 (MACSmix 튜브의 회전을 사용하여 4 RPM)를 품어 ° C. </li><li> gentleMACS의 Dissociator에 C 튜브를 삽입하고 프로그램 &quot;m_brain_02&quot;를 실행합니다. </li><li> 분리 단계 동안 부드럽게 솔루션 솔루션 3 사 10 μl 20 μl를 혼합하여 조직의 400 밀리그램 당 효소 혼합이 30 μl를 준비합니다. </li><li> 심장 - 봉인 캡을 통해 C 튜브에 효소 혼합 2 추가하고 vortexing없이 부드럽게 섞는다. </li><li> 37 C 튜브 10 분에 대한 회전 ° 배양기에서 C를 품어. </li><li> gentleMACS의 Dissociator에 C 튜브를 삽입하고 프로그램 &quot;m_brain_03&quot;를 실행합니다. </li><li> 37 C 튜브 10 분에 대한 회전 ° 배양기에서 C를 품어. </li><li> 튜브의 하단에있는 샘플을 수집하기 위해 간단히 원심 분리기. </li></ol> 3. 여과법 <ol><li> 관심의 세포 통로를 허용하도록 필터 큰만큼을 선택합니다. 예를 들어, Purkinje 세포는 30 μm의 필터를 통과하기에는 너무 큰 있지만, Prominin - 1 + 세포는 것입니다. </li><li> 세포를 제거하는 suitable1000 μl 피펫을 사용 심장 - 봉인 캡을 통해 C 튜브를 형성하고 15 ML 수집 관의 사전 분리 필터를 적용해야합니다. (참고 : 큰 샘플 크기는 50 ML 수집 관을 사용하는 경우). </li><li> HBSS (W) 10 ML과 필터를 씻으십시오. </li><li> RT 10 분 300 XG에서 필터링된 세포를 원심 분리기. </li><li> 후속 응용 프로그램에 대한 중간 또는 선택의 버퍼에 supernatent을 Resuspend. </li><li> myelin의 파편을 제거하려면, Myelin 제거 비즈를 사용합니다. </li></ol> 4. 대표 결과 : 숫자 1-3를 참조하십시오 <img src="/files/ftp_upload/1267/Fig_1.png" alt="그림 1" /> 그림. gentleMACS의 Dissociator 1 뇌 분리는 흐름 cytometric 분석이 보여주는 것처럼, 97% 가능한 세포의 결과. <img src="/files/ftp_upload/1267/1267fig2.png" alt="그림 2" /> 그림. neurosphere 형성 2 라이트 현미경 사진은 자기 세포 정렬 후 맥의 재배 7 일 후 안티 Prominin - 1 MicroBeads를 사용하여 ® NeuroMedium는 맥에서 보충 B27 플러스와 함께 보충. 세포는 신경 조직 분리 키트 (P)를 사용 CD1 마우스 뇌 (P3)에서 준비되었다. <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="무화과 3.5" /> 그림. 단일 셀 suspensions 3 Myelin의 파편이 상당히 세포 절연 impairs 및 Myelin 제거 비즈로 myelin 파편의 제거 효율 전지 분판을 증가시킵니다. 안티 Prominin - 1 MicroBeads을 사용하여 Mac에서 분리 들어, P22 마우스 두뇌 신경 조직 분리 키트 (P)를 사용하여 dissociated되었습니다. 단일 세포 현탁액의 세포가 직접 분리에 사용되었다, 또는 Myelin 제거 비즈를 사용하여 고갈을 myelin에 제출했다. , myelin 제거없이 함께 시료의 분리를 비교하면 순수 이전 myelin 제거와 함께 샘플 높다는 것을 설명합니다.

<ol>
	<li>Hardt, O., Scholz, C., K&#252;sters, D., Yanagawa, Y., Pennartz, S., Cremer, H., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+defines+differences+between+region-specific+GABAergic+neurons.">Gene expression analysis defines differences between region-specific GABAergic neurons.</a> Mol Cell Neurosci. 39, 418-428 (2008).</li><li>Lee, J. K., McCoy, M. K., Harms, A. S., Ruhn, K. A., Gold, S. J., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-protein+signaling+10+promotes+dopaminergic+neuron+survival+via+regulation+of+the+microglial+inflammatory+response.">Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.</a> J Neurosci. 28, 8517-8528 (2008).</li><li>K&#246;rnyei, Z., G&#243;cza, E., R&#252;hl, R., Orsolits, B., V&#246;r&#246;s, E., Szab&#243;, B., V&#225;govits, B., Madar&#225;sz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglia-derived+retinoic+acid+is+a+key+factor+in+glia-induced+neurogenesis.">Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.</a> FASEB J. 21, 2496-2509 (2007).</li><li>Meylan, F., Davidson, T. S., Kahle, E., Kinder, M., Acharya, K., Jankovic, D., Bundoc, V., Hodges, M., Shevach, E. M., Keane-Myers, A., Wang, E. C., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TNF-family+receptor+DR3+is+essential+for+diverse+T+cell-mediated+inflammatory+diseases.">The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.</a> Immunity. 29, 79-89 (2008).</li><li>Skog, J., W&#252;rdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Sena-Esteves, M., Curry, W. T., Carter, B. S., Krichevsky, A. M., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10, 1470-1476 (2008).</li></ol>

Miltenyi Biotec 저자 GmbH를, 독일의 직원입니다.

gentleMACS Dissociator는 닫힌 시스템에서 신경 조직에서 단일 셀 suspensions의 표준 준비를 용이하게합니다. 신경 조직 분리 키트는 immunostainings 및 immunomagnetic 세포 분리 1-5와 같은 추가 응용 프로그램에 필요한 항원 epitopes을 보존하기 위해 최적화되었습니다. 이 프로토콜에서는 97% 가능한 세포에 항복, gentleMACS Dissociator과 신경 조직 분리 키트 (P)를 사용하여 마우스 두뇌의 부드러운 효소 분해를 보여줍니다. 5x10 6 호스트 조직의 연령에 따라 1x10 7 세포 사이에 신경 조직 수익률 100 MG. 대상 Prominin - 1 + 세포는 안티 Prominin - 1 MicroBeads를 사용하여 절연하고 이후 문화로 이동했다. 프로토콜은 쥐&gt; P7에서 파생된 신경 조직과 함께 작업할 때 Myelin 제거 비즈의 추가 사용 훨씬 더 효과적으로 표시됩니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C Tubes</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-237</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>gentleMACS&#x2122; Starting Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-235</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>MACSmix&#x2122; Tube Rotator</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-090-753</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx">http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx</a></td>
</tr>
<tr>
<td>Neural Tissue Dissociation Kit (P)</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-628</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx">http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx</a></td>
</tr>
<tr>
<td>Pre-Separation Filters</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-041-407</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx">http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx</a></td>
</tr>
<tr>
<td>Anti-Prominin-1 MicroBeads</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-333</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx">http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx</a></td>
</tr>
<tr>
<td>Anti-Prominin-1-APC</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-335</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx">http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx</a></td>
</tr>
<tr>
<td>Myelin Removal Beads</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-094-544</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx">http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx</a></td>
</tr>
<tr>
<td>Art 1000 Reach Pipet Tips </td>
<td>&nbsp;</td>
<td>Molecular BioProducts</td>
<td>Order no. 2079</td>
<td><a href="http://www.mbpinc.com/ASP/products.aspx?query_TipID=61">http://www.mbpinc.com/ASP/products.aspx?query_TipID=61</a> 
<a href="http://www.mbpinc.com/html/products/art/display.html">http://www.mbpinc.com/html/products/art/display.html</a></td>
</tr>
<tr>
<td>Neural Tissue Dissociation Kit (T)</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-231</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx">http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx</a></td>
</tr>
<tr>
<td>Propidium Iodide Solution</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-233</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx">http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx</a></td>
</tr>		</tbody>
		</table>
		</div>

generation of single-cell suspensions from mouse neural tissue

신경계 내에의 연결 및 glial 세포 유형의 수백이 설명되었습니다. 뇌 또는 척수의 각 특정 세포 유형은 세포 표면 분자, 또는 그것이 식별하고 특성화 할 수있는을 통해 분자 determinants의 레파토리가 있습니다. 현재, 강력한 세포 식별 및 분리 기술은 동시에 특성 표면 단백질의 세포 죽음과 파괴를 제한하면서 단일 셀 준비가 생성해야합니다. 항원 epitopes을 보존하고 셀 손실을 제한하면서 트립신 또는 papain 기반 분리 키트와 함께 사용 gentleMACS Dissociator는 효과적이고 부드럽게 뇌 조직을 떼어 놓다 수 있습니다. 단일 셀 suspensions의 표준화 준비는 C 튜브 및 최적화, 미리 gentleMACS 프로그램을 사용하여 이루어진다. 일단 생성, 단일 셀 suspensions는 신경 progenitors을 식별하거나, Myelin 제거 비즈를 사용하여 더욱 정화 방지 Prominin - 1 MicroBeads 같은 단클론 conjugates로 치료하실 수 있습니다.

Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

Generation of Single-Cell Suspensions from Mouse Neural Tissue

특정 조직 유형의 세포를 Dissociating하면 조직 aggitation에 대한 특정 매개 변수가 가능한, culturable 세포의 높은 볼륨을 얻기 위해 필요합니다. Miltenyi gentleMACS Dissociator은 간단하고 실용적인 프로토콜이 작업을 최적화합니다. 이 발행물에서 nerual 조직이 장치의 사용이 설명되어 있습니다.

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 ...

generation-of-single-cell-suspensions-from-mouse-neural-tissue

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JoVE Journal

Biology

22.4K 조회수.  Miltenyi Biotec,GmbH.  中文 日本語 

비디오: Generation of Single-Cell Suspensions from Mouse Neural Tissue

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 ...

연구

생물학

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 ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

인간의 사후 뇌 조직에서 단세포 집단의 고품질 RNA 구하기

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

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 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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

마우스와 인간의 대장 조직에서 주요 근육 섬유 모세포의 분리

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

스펙트럼 유동 세포 계측법에 의해 고체 조직에서 분리 한 세포 현탁액의 분석

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Generation of Human Kidney Tubuloids from Tissue and Urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

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 ...