이 작품은 웨스트 버지니아 대학의 메리 Babb 랜돌프 암 센터에서 기금 기금에 의해 지원되었다. 우리는 초기 조언과 도움을 Susette 뮐러 (조지 타운 대학교 (Georgetown University))와 로라 켈리 감사드립니다. 웨스트 버지니아 대학 현미경 영상 시설의 사용은 (메리 Babb 랜돌프 암 센터 및 NIH 보조금 P20 RR16440, P30 RR032138와 P30 GM103488에서 지원) 굉장히 인정하고 있습니다.

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관심 없음 충돌이 선언 없습니다.

세포 외 기질 굴욕 세포를 시각화 할 수있는 능력은 세포 침략의 초기 단계에서 사용 된 분자 메커니즘을 발견에서 도왔습니다. 1980 년대 초 4,14,15에 웬 - 티엔 첸에 의해 개척 이후 미세 분석을위한 유리 coverslips에 코팅 휘황 표시 세포 단백질은 세포 유형의 다양한 범위에 걸쳐 invadopodia 기능을 평가의 기본 기술로 떠오르고있다. 소정의 프로토콜이 일에 비슷한 기존의 형광 및 공 촛점 현미경 11,16-18에서 세포 세포 외 기질 저하의 감지에 적합 미만 2 μm 두께 collagenous 층을 형성 젤라틴 코팅 coverslips를 준비에 사용되는 기본 방법을 보여줍니다 이전에 19-21을 설명하고. 이 속성은 매트릭스 저하의 초기 발병을 감지 할 수있는 코팅 coverslips의 빠른 생산 수 있습니다. 일에 의해 부여 감도전자 전체 매트릭스 환경 (22)의 높은 고유의 강성에 대한 응답으로 invadopodia 형성을 증진 기본 하드 유리 표면 가능성이 보조에 얇은 젤라틴 매트릭스를 가져온다. 그러나, 이러한 매트릭스가 잘 invadopodia 신장 또는 그와 유사한 방법론, 코팅 transwells 또는 전자 현미경 20,23,24과 (30-100 μm) 젤라틴 층을 두껍게을 사용하여 달성 된 추가 형태학의 평가 분석에 적합하지 않습니다. 우리는 사전에 복합 상업적으로 생산 오레곤 그린 488 젤라틴 빠른 실험 세트까지하고 일관성, 재현 결과를 할 수 있습니다 것으로 나타났습니다. 그러나, 라벨이없는 젤라틴에 fluorescein isothiocyanate의 (FITC)의 알칼리성 borate의 활용은 형광 젤라틴 conjugates 20를 생산하기위한 인기있는 저렴한 방법 남아 있습니다. Fibronectin은 라벨과 coverslip 코팅 4,9에 대한 대안 매트릭스 단백질로와의 경우에 사용됩니다vestigators는 고밀도 매트릭스에게 11,25을 만들 라벨이없는 젤라틴 코팅 coverslips에 층을 표시 fibronectin을 사용했습니다. 다른 매트릭스는 세포 유형의 세부 사항에 따라 사용할 수 있습니다. 녹색 488 nm의 스펙트럼의 염료뿐만 아니라, fluorophores의 다양한도 rhodamine 21,26, 알렉사 형석 350 24 546 21 568 5,11 포함한 다양한 형광 스펙트럼과 coverslips을 생성하기 위해 수동 커플 링 방법으로 사용되어왔다 , 594 27 647 5 염료. 이러한 conjugates는 대부분의 모든 이미지 필터 세트에 적합한 특정 ECM 단백질 염색 조합을 활용을위한 유연성을 제공, 소정의 프로토콜에 사용하기 위해 쉽게 적응합니다. 기술은 이전 발표로 본 이기종 인구 또는 전체 셀 그룹에 개별 셀에 귀속 젤라틴 매트릭스 저하를 수량화 할 수 ImageJ를 이용에 필요한 자세한 단계를 제공합니다 설명iously 6,28. 독점 소프트웨어는 성공적으로 같은 목적 5,25 고용되었습니다. 이 프로토콜에서 매트릭스 저하의 영역은 셀의 전체 영역 또는 분야에서 세포의 총 수 (핵) 중 하나에 표준화되어 있습니다. 일반적으로 정규화에 대한 두 옵션은 (ELW, 데이터가 표시되지 않음) 동일한 결과를 제공합니다. 그러나 다른 크기의 셀을 갖는 다른 셀 라인 비교되고 또는 실험적 치료의 크기를 변경하려면 셀을 일으키는 경우,이 휴대폰 번호를 정상화 더 정확 될 수 있습니다. 한편, 많은 암 세포 라인은 총 셀 영역은 정규화에 대한보다 정확한 매개 변수가 될 수있는 경우에, 다중 nucleated 세포의 높은 비율을 갖추고 있습니다. 셀의 일부만이 이미지 (그림 6)에서 캡처하는 경우 또한,이 셀 영역에 정상화보다는 개별 셀의 열화 가능성을 과소 평가하는 것이 더있을 수 있습니다. 그것은 좋은 옷에 이미지 분석을 최적화하는 것이 중요합니다특정 실험 설정의 특성과 뉘앙스. 사람들이 많은 분야에서 전화 번호를 결정하는 경우, 핵을 계산하는 것은 자주 선택하는 방법입니다. ImageJ 자동 계산을위한 핵 카운터 플러그인이 있습니다. 이 도구의 하나 옵션은 분수령 필터입니다. 이 필터는 개별 개체 (그림 5H, 흰색 화살표)로를 segregating하여 수중 별도의 핵을 도움이 될 것입니다. 그러나,이 필터는 (그림 5H, 빨간색 화살표) 광범위하게 겹쳐 별도의 핵 할 수 없습니다. 핵은 불규칙한 모양과 강도의 큰 변화가있는 경우 또한, 필터는 여러 개체 (그림 5H, 노란색 화살표)에 하나의 핵을 분리 할 수 있습니다. 따라서 분석을위한 최적의 매개 변수를 결정하기 위해이 플러그인의 여러 임계 값과 스무딩 방법을 시도하는 것이 중요합니다. 자동 계산 정확한 숫자를 생성하지 않는 경우, 셀 카운터 플러그인은 faci 수<html> 셀 또는 핵의 수동 계산을 litate. 과도 transfection을 이용한 경우, 이미지는 종종 표현 세포의 혼합물을 포함하거나 관심 (그림 6)의 단백질을 표현하지 않습니다. 이 시나리오에서는 세포가 매트릭스 저하의 영역을 만들기위한 책임이있는 항상 명백하지 않습니다. 세포 젤라틴에서 마이그레이션하는 경우 특히 유용합니다. 분석에 일치하도록, 단지 직접 각 셀 아래의 저하 영역을 측정하는 것이 중요합니다. 매트릭스의 어두운 부분을 선택 임계 값과 셀 개요를 생성 할 고를를 사용하여 셀 아래에서만 저하 영역 quantitated 될 것입니다. 이 절차는 분석에서 셀 경계 (그림 6 층, 화살표) 이외의 저하 지역을 제외합니다. 분석은 세포가 이동하는 기회가 전에 저하위한 충분한 시간을 허용하는 시점을 선택 최적화가 필요할 수 있습니다. &quot;&gt; 많은 방법이 invadopodia 형성과 기능을 quantitate하기 위해 개발되었습니다. 매트릭스 저하뿐만 아니라, 다른 자주보고되는 매개 변수는 셀 당 invadopodia의 수, 주어진 인구에서 invadopodia를 표시하는 세포의 비율과 수를 결정하는 등 &#39;미숙을 &quot;또는&quot;사전 저하 ECM 11,13,25,26 수있는 성숙 &#39;invadopodia &quot;비 굴욕 invadopodia는 비교&quot;. invadopodia 평가를위한 선택의 방법 (들) 각 셀 형식의 고유 특성에 따라 달라집니다. 예를 들어, 셀 당 invadopodia의 수를 계산하거나 invadopodia를 포함하는 세포의 비율을 결정하는 것은 분석 세포가 몇 눈에 띄는 invadopodia가 포함 된 경우 잘 작동하는 간단한 방법이지만, invadopodia 수십 또는 위치를 invadopodia 작은 수 있습니다이 세포에 더 어려워지고 어려운 감지합니다. 열화 분석을 사용하면이 가능 사전 invadopodia V의 비율을 계산 할 수 있습니다s입니다. 저하 매트릭스있는 비율 invadopodia과 세포의 총 수를 비교하여 단일 세포 또는 인구의 성숙 invadopodia. 적은 세포가 invadopodia를 표시 세포에 비해 저하 매트릭스 아르 차이가있는 경우, 이러한 세포는 세포가 해결 된 시간에 매트릭스 저하 수없는있었습니다 사전 invadopodia을 형성하고 있음을 나타냅니다 할 수 있습니다. 분석을 위해 선택되어이든 방법을 조합, 그건은 객관적으로 가능한 한 원하는 invadopodia 특성을 수량화하는 것이 중요합니다. 현미경에 이미지를 수집 할 때 우선적으로 저하 높은 수준의와 지역을 선택할 편견을 피하기 위해 오히려 형광 매트릭스보다 세포 (고를),보고 필드를 선택합니다. 다중 이미지는 세포 인구의 공정한 표현을 보장하기 위해 취득해야합니다. 이미지는 적절한 배율로 취득해야합니다. 세포의 균일 한 인구를 들어, 낮은 배율 수저하 등의 분야는 여전히 해결 될 수하는 한 더 많은 세포를 수집하는 데 사용할 수. 높은 배율 이미지는 개별 셀에서 영역을 측정하고 개별 invadopodia를 해결​​하는 데 좋습니다. 영역이 quantitated되는 경우, 강도에 따라 이미지를 임계 값은 수동으로 측정 할 수있는 매트릭스의 영역을 선택보다 더 객관적이다. 모든 경우에, 여러 독립적 인 실험에서 세포의 충분한 숫자 통계적으로 의미, 재현 결과를 제공하는 분석해야합니다.

<table border="1"><tbody><tr><td> 시약 / 악기의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 젤라틴 </td><td> 시그마 </td><td> G1890 </td><td> 돼지 피부 </td></tr><tr><td> 자당 </td><td> 어부 </td><td> BP220 </td><td></td></tr><tr><td> 12mm coverslips </td><td> 어부 </td><td> 50-121-5159 </td><td></td></tr><tr><td> 24 잘 접시 </td><td> 어부 </td><td> 08-772-1 </td><td> BD 팔콘 </td></tr><tr><td> 질산 </td><td> Ricca </td><td> R5326000 </td><td> 20 % 솔루션 </td></tr><tr><td> 폴리-L-라이신 </td><td> 전자 현미경 과학 </td><td> 19320-B </td><td> 0.1 % 용액 </td></tr><tr><td> 글 루타 알데히드 </td><td> 시그마 </td><td> G7526 </td><td> 8 % 솔루션 </td></tr><tr><td&gt; 오레곤 그린 488 - 복합 젤라틴 </td><td> Invitrogen </td><td> G-13186 </td><td></td></tr><tr><td> 나트륨 borohydride </td><td> 시그마 </td><td> 213462 </td><td></td></tr><tr><td> 트리톤 X-100 </td><td> 어부 </td><td> BP151 </td><td></td></tr><tr><td> rhodamine-phalloidin </td><td> Invitrogen </td><td> R415 </td><td></td></tr><tr><td> 반 cortactin (클론 4F11) </td><td> Millipore </td><td> 05-180 </td><td></td></tr><tr><td> 안티 - 플래그 항체 </td><td> Millipore </td><td> MAB3118 </td><td></td></tr><tr><td> 알렉사 형석 647 염소 안티 - 마우스 IgG </td><td> Invitrogen </td><td> A21235 </td><td></td></tr><tr><td> 골드 antifade을 연장 </td><td> Invitrogen </td><td> P36930 </td><td></td></tr><tr><td> LSM 510 공 촛점 현미경 </td><td> Zeiss </td><td></td><td></td></tr><tr><td> ImageJ 소프트웨어 </td><td> 공개 </td><td></td><td> <a href="http://www.macbiophotonics.ca/imagej/" target="_blank">http://www.macbiophotonics.ca/imagej/</a> </td></tr><tr><td> AutoQuant X2.2 소프트웨어 </td><td> 미디어 인공 이식 </td><td></td><td></td></tr><tr><td> NIS 요소 소프트웨어 </td><td> 니콘 </td><td></td><td></td></tr></tbody></table>

quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts

지역 조직에 세포 침공은 개발과 항상성에서 중요한 과정입니다. Malregulated 침공과 이후의 세포 운동은 염증, 심장 혈관 질환과 종양 세포 전이 한 등 여러 가지 병적 인 프로세스의 특징입니다. 상피 또는 내피 지하 막에서 세포 외 기질 (ECM)의 구성 요소 Focalized proteolytic 저하는 세포 침공을 시작으로 중요한 단계입니다. 종양 세포에서 체외 분석에 광범위한는 ECM 저하가 invadopodia 2,3를 칭했다 복부 고를 풍부한 막 밀어내는 구조에 의해 수행되는 결정을 내 렸습니다. 그들은 매트릭스 metalloproteinases (MMPs)의 행동을 통해 ECM 분석을 검토 ECM에 가까운 동격의 Invadopodia 양식. invadopodia을 형성하는 종양 세포의 능력은 바로 지역의 기질 및 관련 혈관 구성 요소를 3으로 침입 할 수있는 능력과 함께 연결합니다. _content는 &quot;유리 coverslips에 코팅 염료 표시 매트릭스 단백질을 사용하여 형광 현미경에 의한 세포의 invadopodia로 인한 ECM 저하의&gt; 시각화는 매트릭스 proteolysis 및 셀룰러 침해 가능성 4,5의 정도를 평가하기위한 가장 널리 기술로 떠오르고있다. 여기에 언급 된 상업적으로 이용 가능한 오레곤 그린 - 488 젤라틴의 공액을 활용 휘황 라벨이 표시된 유리 coverslips을 생성하기위한 표준 방법의 버전입니다.이 방법은 쉽게 빠르게 코팅 coverslips 많은 수의 생산 조정됩니다. 우리는 흔히 발생하는 일반적인 미세 유물의 일부를 보여 이 절차를 수행하는 동안 얼마나 이것들은 방지 할 수 있습니다. 마지막으로, 우리는 개별 셀 의해 전체 휴대폰 인구에 의해 중재 표시 젤라틴 매트릭스 저하의 정량화 할 수 있도록 즉시 사용할 수 컴퓨터 소프트웨어를 사용하여 표준화 된 방법을 설명합니다. 설명 절차는 정확하고 reproducibly 모니터링 할 수있는 기능을 제공 invadopodia활동, 또한 변조 단백질의 발현 또는 단일 및 다세포 설정에서 세포 외 기질 저하에 반 침해 화합물의 테스트의 효능을 평가하기위한 플랫폼으로 제공 할 수 있습니다.

Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

우리는 invadopodia로 인한 매트릭스 저하를 시각화를위한 형광 젤라틴으로 코팅 현미경 coverslips를 생산을위한 원형 방법을 설명합니다. 사용 가능한 소프트웨어를 사용하여 전산 기술이 혼합 된 인구 내에 전체 미세 필드를 포함한 다세포 그룹에 대한 하나의 세포 매트릭스 proteolysis의 결과 수준을 정량화를 위해 제공됩니다.

단일 및 다세포 상황에서 Invadopodia로 인한 세포 외 기질 Proteolysis의 양적 측정

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

quantitative-measurement-invadopodia-mediated-extracellular-matrix

Nanyang Technological University

Research

JoVE Journal

Biology

19.2K 조회수.  West Virginia University. 우리는 invadopodia로 인한 매트릭스 저하를 시각화를위한 형광 젤라틴으로 코팅 현미경 coverslips를 생산을위한 원형 방법을 설명합니다. 사용 가능한 소프트웨어를 사용하여 전산 기술이 혼합 된 인구 내에 전체 미세 필드를 포함한 다세포 그룹에 대한 하나의 세포 매트릭스 proteolysis의 결과 수준을 정량화를 위해 제공됩니다.

비디오: Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

유동세포계측법에 의해 플라즈마 막에 GLUT4 Translocation의 양적 측정

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

연구

생물학

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

전체 세포 인구의 단일 셀 수준에서 Cyclin B의 Proteolysis 유학

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

프로브 기반의 실시간 PCR은 마이크로 RNA의 정량 측정을위한 접근 방법

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

효율적인 포유 동물 세포 발현 및 결정화에 대한 세포 외 당 단백질의 단일 단계 정화

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

침연 (macerated) 조직의 전자 현미경을 스캔하면 세포 외 매트릭스를 시각화하기

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

세포 유래 세포 외 매트릭스의 빠른, 분리, 기능 연구에 대한 확장 방법, 및 분석

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

마이크로 유체 장치를 사용하면 단일 신진 효모 세포의 수명과 세포 표현형을 측정하는

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

세포 외 기질 식 및 선 형태 분석을 그대로, 전체 마우스 유 방 땀 샘의 절연

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...