저자는 murine Ngn3 - EGFP 배아 줄기 세포 라인 부인, 루시 브라운과 셀 정렬에 도움에 대한 분석 Cytometry 코어의 선물 박사 클라우스 Kaestner 감사하고 싶습니다. 이 작품은 MW와 LS에게 수여 훈련 보조금 HTK에게 수여 NIH 보조금 R21DK069997와 R01DK081587에 의해 및 재생 의료 (CIRM)의 캘리포니아 연구소에서 일부 재정 지원되었다

<ol>
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우리는 공개 아무것도 없어.

개별 췌장 progenitors의 양적 및 기능적 평가를 허용 식민지의 assays는 크게 췌장 줄기 및 전구 세포 생물학의 연구에 진전을 방해하는, 지금까지 제한하고 있습니다. 식민지 분석의 본질은 전구 세포의 기능과 혈통의 잠재력을 관찰 수 있도록, 내장 전구 세포의 능력보다는 전적으로 자신의 표현형을 측정하는 것입니다. 또한 주어진 인구 progenitors의 수를 결정할 수 있습니다 양적 도구입니다. 조혈 줄기 세포 분야에서 식민지의 assays는 식별에 중요한 역할을뿐만 아니라, 이러한 세포의 혈통의 의지를 지배하는 성장 인자 요구 사항, 상호 작용, 그리고 메커니즘을 해독했습니다. 6 췌장 전구 세포 생물학, 췌장 장기 7 입력란에 , 8 또는 일괄 문화 9-13 고안되었습니다 그러나, 이러한 assays는 생물 학적 questi를 해결하지단일 세포 수준에서 기능. 단일 세포 분석없이, 다중 잠재적인 췌장 줄기 / progenitors의 존재는 명백하게 입증되지 않을 수 있습니다. 14 여기는 것을 보여줄 인슐린 표현 식민지 - 형성 progenitors 것은 3 차원, Matrigel 함유, 세미 솔리드 문화 시스템을 사용 assayed 수 있습니다. 서스펜션 문화 시스템 15,16가 분아 따위에 의해 중식 및 &quot;pancreatospheres&quot;로 차별화하는 데 하나의 성인 췌장 progenitors를 허용하는 개발되었습니다 있지만, 우리의 식민지 분석 추가 이점을 제공합니다. 첫째, 우리 세미 솔리드 문화 미디어 시스템은 최대 24 자 당 500 μL 문화 미디어 당 25,000 정렬 세포로의 도금 선형 범위의 결과 식민지 형성을 유지하면서. 2 대조적으로, 서스펜션 pancreatosphere 분석은 하나의 사용을 필요로 수 세포 분류기에서 전지 증착 방법은 단일 세포 분석을 허용하고, 따라서 문화 매체와 인큐베이터 공간의 큰 볼륨으로이 필요합니다. 따라서, 우리의 식민지 분석은 다른 세포의 다수가 전구 활동을 동시에 비교해야 할 때 실시하는 비교적 쉽고 비용 효과적입니다. 둘째, 우리 세미 솔리드 미디어 세포외 기질 구성 요소 및 증식과 분화가 모두 같은 잘 발생 수 성장 요인의 균일한 분포를하실 수 있습니다. 반대로, 그것은 서스펜션 문화에 세포 이러한 환경, 특히 매트릭스를 제공하기가 더 어렵습니다. 두 가지 progenitors가 무작위로 서로 양식을 한 식민지 근처에 위치 수 있으므로 우리의 방법으로 그것이 정말 로요, 각 식민지가 세포 하나에서 파생되는 것을 증명 수 없다는 것을 알고 있습니다. 그러나이 문제는 단일 세포 조작을 사용하여 보조 분석에 의해 극복할 수 있습니다. 정렬 단일 세포를 직접 손으로 따기가 정말 로요 각 식민지의 단세포 출처를 보여줍 수 있습니다. 요약에서 프로의 식민지 - 성형 활동을 인슐린을 표현반 고체 미디어 genitors 언급된 단일 세포 수준에서 췌장 progenitors의 기계론의 연구에 대한 독특한 기회를 제공합니다 설명했다. 예를 들어, 이전 보고서에서 우리는 문화에 미디어가 인슐린을 표현 세포외 매트릭스를 제안 Ngn3 - EGFP + 세포, 2 식민지 것은 형성에 필요한, Matrigel, 세포외 기질 단백질의 원유 혼합물의 추가 발견 그 progenitors 위해 중요합니다. 이 식민지 분석을 사용하여 이제 더 이상 해부하다와 같은 활동에 대한 구체적인 매트릭스 요구 사항을 정의하는 것이 가능합니다. 마우스 배아 줄기 세포에서 파생된 progenitors를 공부하는 것 외에도, 우리는 현재이 시험 관내 분석 시스템은 다른 독특한 perinatal 성인 생쥐의 췌장과 간장에서 파생된 식민지뿐만 아니라, 결여된 인간 시체 같은 췌장 조직의 형성을 지원하는지 여부를 조사하고 있습니다 islets니다.

<table border="1"><tbody><tr><td> 재료 / 시약 </td><td> 회사 </td><td> 카탈로그 수 </td><td> 댓글 </td></tr><tr><td> 1X DMEM / F - 12 (1:1) </td><td> Mediatech </td><td> 15-090 - 이력서 </td><td></td></tr><tr><td> 소태아 혈청 </td><td> 조직 문화 체액 </td><td> 201 </td><td> 섹션을 참조하십시오 1.1 </td></tr><tr><td> L - 글루타민 </td><td> Gibco </td><td> 25030-081 </td><td></td></tr><tr><td> 1X Dulbecco의 PBS </td><td> Mediatech </td><td> 21-031 - 이력서 </td><td></td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> A8412 </td><td> 7.5 % </td></tr><tr><td> Matrigel </td><td> Becton 디킨슨 </td><td> 356,231 </td><td> 감소 성장 인자 </td></tr><tr><td> 메틸 셀룰로오스 </td><td> Sinetsu 화학, 도쿄, 일본 </td><td></td><td> 1500 centipoise (높은 바이올렛scosity) </td></tr><tr><td> 니코틴 </td><td> 시그마 </td><td> N0636 </td><td></td></tr><tr><td> 사람의 재조합 Activin βB </td><td> R &amp; D 시스템 </td><td> 659 - AB - 025 </td><td></td></tr><tr><td> Exendin - 4 </td><td> 시그마 </td><td> E7144 </td><td></td></tr><tr><td> 사람의 재조합 VEGF - A </td><td> R &amp; D 시스템 </td><td> 293 - VE - 010 </td><td></td></tr><tr><td> 24 잘 접시 </td><td> Costar </td><td> 3473 </td><td> 초저 부착 </td></tr><tr><td> 60mm 페트리 접시 그리드와 함께 </td><td> Nunc </td><td> 169,558 </td><td></td></tr></tbody></table>

a quantitative assay for insulin-expressing colony-forming progenitors

췌장 줄기 및 전구 세포 생물학 분야는 단일 세포의 분석을 위해 허용 체외 기능 및 정량 assays에의 부족에 의해 방해되었다. 그들은 명백하게 개별 progenitors의 혈통 잠재력을 증명하는 확실한 방법을 제공하기 때문에 단일 progenitors의 분석 매우 중요 있습니다. 방법은 하나의 세포에서 서스펜션 문화 &quot;pancreatospheres&quot;을 생성 고안되어 있지만, 몇 가지 제한이 존재합니다. 첫째, 단일 셀 전지의 대규모 번호를 증착, 회전 명령의 문화 매체와 공간의 대량.을 수행하려면 시간이 많이 소요됩니다 결과 pancreatospheres의 둘째, 세는 법은 pancreatosphere - 시작 progenitors의 빈도가 낮은 특히, 노동 집약이다. 셋째, pancreatosphere 분석을 다시 같은 문화의 확산과 췌장 progenitors의 차별이 모두 잘 수 있도록 효율적인 방법은 아닙니다분석의 유용성을 stricting. 이러한 한계를 극복하기 위해 췌장 progenitors을위한 세미 솔리드 미디어 기반의 식민지 분석이 개발되었고이 보고서에 표시됩니다. 이 방법은 그들이 확산뿐만 아니라 차별을 받다으로 전구 세포가 3 차원 공간에 머물 수 있도록 메틸 셀룰로오스가 언론에 점도를 제공하는 데 사용되는 조혈 식민지 분석에서 기존의 개념을 활용합니다. 풍성하게하기 위해 인슐린을 표현하는 이질적인 인구에서 progenitors을 식민지를 형성, 우리는 세포를 이용 것을 표현 neurogenin (NGN) 3, 췌장 내분비 전구 세포 마커. Murine 배아 줄기 (ES)는 향상된 녹색 형광 단백질 기자와 태그 Ngn3 표현 세포가 정렬되었습니다 잘 따라 많은 25,000으로 세포가 낮은 첨부 24 - 물론 문화 요리에 도금되었다 셀 - 파생. 다음과 같은 주요 구성 요소와 세미 솔리드 미디어의 각 잘 포함된 500 μL가 : 만났다hylcellulose, Matrigel, 니코틴, exendin - 4, activin βB 및 시설 언론 murine ES 세포 - 유래 췌장 같은 세포에서 수집했습니다. 문화의 8~12일 후, 인슐린은 - 표현 특유의 형태로 식민지 것은 형성되었으며 추가로 각 식민지의 혈통 구성을 결정하는 정량 RT - PCR과 immunoflourescent 염색법을 사용하여 췌장 유전자 표현 분석 수 있습니다. 요약 우리의 식민지 분석 쉽게 감지 및 세포의 이질적인 인구 내에서 기능 progenitors의 부량 수 있습니다. 또한, 반 고체 미디어 형식은 체외에서 세포 분열 따위에 의해 번식하고 차별화하는 데 progenitors을 활성화, 세포 세포외 기질 구성 요소와 성장 요인 유니폼 프레 젠 테이션을하실 수 있습니다. 이 식민지 분석은 단일 세포 수준에서 췌장 전구 세포의 기계론의 연구에 대한 독특한 기회를 제공합니다.

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A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

췌장과 같은 progenitors은 인슐린을 표현 식민지로 차별화할 수있는 3 차원 clonogenic 분석이 설명되어 있습니다. 이 방법은 메틸 셀룰로오스, Matrigel 및 성장 요인을 포함하는 반 고체의 미디어 활용,하는 하나의 progenitors는 분아 따위에 의해 중식과 차별<em&gt; 체외에서</em&gt; 인구의 기능 progenitors의 수를 부량를 허용.

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

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Biology

13.5K 조회수.  California State University Channel Islands. 췌장과 같은 progenitors은 인슐린을 표현 식민지로 차별화할 수있는 3 차원 clonogenic 분석이 설명되어 있습니다. 이 방법은 메틸 셀룰로오스, Matrigel 및 성장 요인을 포함하는 반 고체의 미디어 활용,하는 하나의 progenitors는 분아 따위에 의해 중식과 차별 체외에서 인구의 기능 progenitors의 수를 부량를 허용.

비디오: A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

연구

생물학

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

CD71/TER119 흐름 cytometric 어세를 사용하여 마우스 Erythroid Progenitors의 제품 식별 및 분석

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

내피 콜로니 형성 세포의 Phenotypic 및 기능성 특성은 인간 탯줄의 혈액에서 유래

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

양적 휘트니스 분석 워크플로우

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

민감한, 대규모 양적 대사 체학을위한 전략

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

정량 면역 형광 분석은 중심체에서 단백질 수준의 변화를 측정하기 위해

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

CD71/TER119 흐름 cytometric 어세를 사용하여 마우스 Erythroid Progenitors의 제품 식별 및 분석

내피 콜로니 형성 세포의 Phenotypic 및 기능성 특성은 인간 탯줄의 혈액에서 유래

양적 휘트니스 분석 워크플로우

펩티드 에피토프의 정량 분석을위한 높은 처리량 MHC II 결합 분석

민감한, 대규모 양적 대사 체학을위한 전략

정량 면역 형광 분석은 중심체에서 단백질 수준의 변화를 측정하기 위해

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

CD71/TER119 흐름 cytometric 어세를 사용하여 마우스 Erythroid Progenitors의 제품 식별 및 분석

내피 콜로니 형성 세포의 Phenotypic 및 기능성 특성은 인간 탯줄의 혈액에서 유래

양적 휘트니스 분석 워크플로우

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

민감한, 대규모 양적 대사 체학을위한 전략

정량 면역 형광 분석은 중심체에서 단백질 수준의 변화를 측정하기 위해

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

펩티드 에피토프의 정량 분석을위한 높은 처리량 MHC II 결합 분석