우리 작품은 NIH 보조금 NS048447 및 AG017216to KD에 의해 지원됩니다. 리 리우는 원래 프로토콜의 개발 기간 동안 자신의 감독 및 지원에 대한 박사 Heikki Tanila을 (쿠오 피오 대학, 쿠오 피오, 핀란드) 감사드립니다.

유리 모세관 튜브를 풀링 <ol><li> 유리 모세관은 셔터 악기 부동산 (Borosilicate 유리, B100 - 75 - 10)에서 구입할 수 있습니다. </li><li> 서터에서 P - 87 300 세트 열을 색인과 330에 설정된 압력 지수와 micropipette 풀러 플라밍. 모세관 튜브를 당겨 </li><li> 테이퍼 팁에 대해 0.5 mm의 내경을 가지고 있도록, 가위로 유리 모세관 튜브의 끝부분을 낸다. </li></ol> CSF에 대한 Cisterna 마그나 찔린 기술 CSF 샘플은 cisterna 마그나 (그림 1) 이전에 한 출판 되었음 방법을 사용에서 가져옵니다. <img src="/files/ftp_upload/old/960_2008_10_13_2/960_Figure-01.png" alt="그림 1" width="432" height="184" /> 그림 1 1. 생쥐에 마취제 (100mg/kg)과 xylazine (10mg/kg), 관리 intraperitoneally에 의해 anesthesized 있습니다. 마취 유도의 기간 동안, 생쥐는 37 O C 인큐베이터에 보관됩니다. 2. 목 피부는 면도 있으며, 마우스는 다음 손난로의 직접적인 접촉과 stereotaxic 악기에 경향이 배치됩니다. 가열 패드에 의해 생산되는 열을 체온의 변화에​​ 대응 조정되도록 직장 온도 프로브는 항문에 삽입됩니다. 머리는 머리 어댑터 보안입니다. 외과 사이트는 70 %의 에탄올 (3 번 반복) 다음, 10 % povidone의 요오드로 채취하고, 피부의 화살 절개는 후두에 열등 이루어집니다. 3. 해부 현미경 아래 피하 조직과 근육이 (M. biventer cervicis 및 M. rectus capitis 발등 전공) 포셉과 무딘 절개로 구분됩니다. microretractors의 두 사람이 떨어져 근육을 보유하는 데 사용됩니다. 4. 마우스는 머리가 시체와 함께 거의 135 O 각도를 형성 있도록 누웠지입니다. 5. 해부 현미경 아래에서 cisterna 마그나의 두라 메이터은 모수의 oblongata 및 주요 혈관 (arteria 발등 spinalis) 및 CSF의 공간 (그림 2) 표시되는를 통해 반짝 이는 맑은 역방향 삼각형으로 나타납니다. <img src="/files/ftp_upload/old/960_2008_10_13_3/960_Figure-02.png" alt="그림 2" width="304" height="307" /> 그림 2 6. 두라 메이터는 멸균 면봉과 건조 blotted 있습니다. arteria 발등 spinalis (그림 2) 측면 두라 메이터를 통해 citerna 마그나에 모세관에 침투. 모세관 튜브 삽입에 저항에 눈에 띄게 변화에 따라, CSF는 모세관으로 흐르고 있습니다. 7. 조심스럽게 모세관 튜브를 제거하고 1mm 내경을 가진 폴리에틸렌 튜브를 통해 3 ML의 주사기에 연결합니다. 미리 표시 0.5 ML eppendorf 튜브에 CSF를 주사하고, 드라이 아이스에 즉시 튜브를 동결하고 -80 ° C 냉동고에 그것을 전송합니다. 8. CSF 샘플링 후, 근육이 다시 정렬하며, 피부가 (4-0, Ethicon, 존슨 앤 존슨) 봉합합니다. 0.9 % NaCl의 약 1 ML은 드 수화를 방지하기 위해 subcutaneously 주입니다. 마우스가 복구까지 체온을 유지하기 위해 인큐베이터에 보관되며 마우스의 무게는 1 일, 그리고 수술 후 일주를 감시하고 있습니다. 결과 및 토론 우리는 감지 플라즈마 오염없이 마우스에서 CSF의 시리얼 샘플링에 대한 신뢰성이 높은 프로토콜을 설명했다. 1. 전체 절차는 일반적으로 마우스를 따라 10 분 (마취 포함) 정도 소요됩니다. CSF 획득의 볼륨 마우스 변종에 따라 좌우됩니다. P301L (JNPL3) 마우스 3 10 μl - 15 μl의 수율을 제공하면서 2를 사용하여 PS / APP 더블 TG 생쥐에서 평균 거래량이 5 μl (3-7 μl)입니다. 시리얼 샘플링의 경우, 7-8 μl의 최대 안전 2~3개월의 간격으로 각각의 시간을 촬영하실 수 있습니다. 2. 수술하는 동안, 그것은 cisterna 마그나의 두라 메이터이 충분히 노출 수 있도록 stereotaxic 프레임에서 제대로 마우스의 머리와 신체를 위치하는 것이 중요합니다. ApoB 4 immunoblotting하여 CSF 샘플에서 모니터링할 수 있습니다 플라즈마 단백질에서 오염을 방지하기 위해 모세관으로 두라 메이터을 관통 때 신중하게 혈관을 피하십시오. 3. 조직의 교착은 다음 샘플링을 위해 predisposing 출혈로 어려움을 높일 수있는 각각의 수술시 조직 손상을 최소화하는 것이 중요합니다. 4. 마취로 인한 저체온증은 상당히 매우 빠르게 5 두뇌에 phosphorylated 타우 수준에 영향을 미칠 수 있기 때문에 체온이 잘 수술하는 동안 유지되어야합니다. 5. 우리가 사용하는 AB 엘리사 프로토콜과 항체는 Refolo 외에 자세히 설명했다. 6. 1:50-1:60 희석 때 PS / APP 생쥐에서 CSF의 두 μl는 만족스러운 결과를 줄 것이다. 타우 엘리사 들어, P301L (JNPL3) 생쥐 4-5 μl CSF는 충분합니다총 타우, 또는 타우의 검출 인광 물질은 트레오닌에서 ylated 231 (키트는 바이오 - 소스에서 구입). 6. 이 기술은 CSF의 작은 볼륨이 원하는 assays에 대해 만족하는 대한 신경 질환의 다른 마우스 모델에 적용할 수 있습니다.

<ol>
	<li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observation+on+CSF+Abeta42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF Abeta42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol Dis. 17, 516 (2004).</li><li>Holcomb, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+Alzheimer-type+phenotype+in+transgenic+mice+carrying+both+mutant+amyloid+precursor+protein+and+presenilin+1+transgenes.">Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes.</a> Nat Med. 4, 97 (1998).</li><li>Lewis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibrillary+tangles,+amyotrophy+and+progressive+motor+disturbance+in+mice+expressing+mutant+(P301L)+tau+protein.">Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.</a> Nat Genet. 25, 402 (2000).</li><li>DeMattos, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plaque-associated+disruption+of+CSF+and+plasma+amyloid-beta+(Abeta)+equilibrium+in+a+mouse+model+of+Alzheimer's+disease.">Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease.</a> J Neurochem. 81, 229 (2002).</li><li>Planel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+leads+to+tau+hyperphosphorylation+through+inhibition+of+phosphatase+activity+by+hypothermia.">Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.</a> J Neurosci. 27, 3090 (2007).</li><li>Refolo, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cholesterol-lowering+drug+reduces+beta-amyloid+pathology+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease.</a> Neurobiol Dis. 8, 890 (2001).</li></ol>

우리는 감지 플라즈마 오염없이 마우스에서 CSF의 시리얼 샘플링에 대한 신뢰성이 높은 프로토콜을 설명했다. 1. 전체 절차는 일반적으로 마우스를 따라 10 분 (마취 포함) 정도 소요됩니다. CSF 획득의 볼륨 마우스 변종에 따라 좌우됩니다. P301L (JNPL3) 마우스 3 μl - 15 10 μl의 수율을 제공하면서 우리가 used2 PS / APP 더블 TG 생쥐에서 평균 거래량이 5 μl (3-7 μl)입니다. 시리얼 샘플링의 경우, 7-8 μl의 최대 안전 2~3...

a technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse 

알츠하이머 병 (AD)는 병적 β - 아밀로이드 펩티드 (Aβ)와 hyperphosphorylated 타우 단백질의 세포외 intraneuronal 축적의 증착에 의해 특징 진보적인 neurodegenerative 질병입니다. 중추 신경계 (CSF)는 두뇌의 세포외 공간에 직접 접촉하기 때문에, 그것은 병적인 과정에 대한 응답으로 뇌의 생화학 변화의 반영을 제공합니다. 이러한 변화에 책임이있는 메커니즘이 아직 완전히 이해되지 않습니다하지만 AD 환자의 CSF는 42 아미노 산성 Aβ (Aβ42)의 형태, 총 타우와 타우 hy​​perphosphorylated 증가에서 감소를 보여줍니다. AD의 형질 (TG) 마우스 모델을 어떻게 그리고 왜 Aβ 또는 타우 수준 CSF의 변화에​​ 질병이 진행됨에 따라 조사에 좋은 기회를 제공합니다. 여기, 우리는 마우스 CSF 샘플링을위한 세련된 cisterna 마그나 찔린 기법을 보여줍니다. 이것은 매우 부드러운 샘플링 기술은 시리얼 CSF 샘플이 크게 시간이 지남에 따라 미묘한 변경을 감지하는 것이 가능하게, Aβ 또는 타우 수준 사이 마우스 변화의 혼란함을 주죠 효과를 최소화 2~3개월 간격으로 같은 마우스에서 구할 수 있습니다. Aβ 및 타우 엘리사와 함께이 기술은 AD 마우스 모델에서 뇌의 CSF Aβ42과 타우의 수준 사이의 관계, 그들의 신진 대사를 조사하기위한 연구에 유용합니다. TG 생쥐의 연구 모니터링 질병 진행에 대한 생물 학적 마커로 사용할 수 CSF의 Aβ 또는 타우 수준의 잠재적으로 중요한 검증을 제공할 수있다, 그리고 치료 개입의 효과를 모니터합니다. 생쥐가 희생될 수 있고 두뇌가 생화학 또는 histological 변경 검사를 할 수 있듯이, CSF 변경 기본 메커니즘은 더 나은 평가하실 수 있습니다. 이 데이터는 인간의 광고 CSF 변경의 해석에 대한 정보를 수있다.

Watch this Scientific Journal Video about A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse at JoVE.com

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

AD의 형질 (TG) 마우스 모델을 어떻게 그리고 왜 Aβ 또는 타우 수준 CSF의 변화에​​ 질병이 인간의 환자에서 진행됨에 따라 조사를 위해 좋은 기회를 제공합니다. 여기, 우리는 마우스 시리얼 CSF 샘플링을위한 세련된 cisterna 마그나 찔린 기법을 보여줍니다.

Cisterna 마그나 마우스에서 뇌척수의 직렬 그룹에 대한 기술

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

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A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

100.7K 조회수.  Columbia University.  AD의 형질 (TG) 마우스 모델을 어떻게 그리고 왜 Aβ 또는 타우 수준 CSF의 변화에​​ 질병이 인간의 환자에서 진행됨에 따라 조사를 위해 좋은 기회를 제공합니다. 여기, 우리는 마우스 시리얼 CSF 샘플링을위한 세련된 cisterna 마그나 찔린 기법을 보여줍니다. 

비디오: A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

에서 세포 외액의 Vampiric 절연<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

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생물학

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 ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

장기 성인 마우스 SSC 라인의 유도를위한 문화 Spermatogonial 줄기 및 전구 세포의 일련 농축 (SSCs)

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

마우스 췌장에서 RNA 격리 : 리보 뉴 클레아 풍부한 조직

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

공장에서 유체 Apoplastic의 추출을위한 침투-원심 분리 기술을 사용하여 잎<em&gt; 위 Phaseolus 심상</em&gt; 예를 들어

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

총회와 &#39;전단 반지&#39;의 응용 프로그램 : 궤도, 단방향 및 정기 유체 흐름 및 전단 응력에 대한 소설 내피 모델

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

마우스입니다 관류 신장 기법

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

페리 이식 단계 인간 배아에서 트로포 블라스트 세포의 단일 세포 수집

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

성인 마우스 입천장에서 1 차 구강 각막 세포의 격리 및 문화

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.