우리는 세포 라인을위한 시약 및 프레드 밀러 (바바라 앤 Karmanos 암 Intitute, 디트로이트, 미국)의 켄 이와타 (OSI 제약, 뉴욕, 미국)에게 감사하고 있습니다.

1. methocel 솔루션의 준비 (500 ML) <ol><li> 자기 활동가를 포함한 500 ML 병에 메틸 셀룰로오스의 압력솥 6g. </li><li> 20 분에 대한 혈청 (60 ° C)없이 preheated DMEM/F12 250 ML에 메틸 셀룰로오스를 분해. </li><li> 10 % 말 혈청을 포함하는 DMEM/F12 (RT) 250 ML을 추가합니다. 믹스 하룻밤 (4 ° C). </li><li> 원심 분리 (5천그램, 2 시간, 4 ° C)의 솔루션을 취소합니다. </li><li> 새로운 튜브 (재고 솔루션의 9​​0-95 % 정도)에 명확한 높은 점성 솔루션을 가져가라. </li><li> 4 저장 ° C까지 사용. </li></ol> 2. 회전 타원체 문화 <ol><li> 20 % methocel 솔루션 (10 ML methocel 40 ML 성장 매체) 준비. </li><li> PBS로 두 번 세포를 씻어, 37에서 0.1 %의 트립신과 부화 세포를 추가 ° C </li><li> 성장 매체 세포를 Resuspend하고 세포를 계산합니다. </li><li> 20 % methocel에서 ML 당 10 000 세포 현탁액을 준비합니다. </li><li> 96 잘 둥근 바닥의에 세포 현탁액을 추가uspension 플레이트 (물론 당 100 μl). </li><li> 다음날, 서스펜션 플레이트의 회전 타원체 형성을 확인합니다. Spheroids 1-3 일 후에 사용할 준비가됩니다. </li></ol> 3. 콜라겐의 준비 <ol><li> 얼음 8 ML의 콜라겐, 1 ML 10X PBS, 1 ML 0.1 N NaOH 3 방울 0.1 N HCL의 솔루션을 준비합니다. 산도가 산도 표시기 스트립에있는 솔루션의 일부를 야생으로 7.4 있는지 확인하십시오. 인지 범위의 산도를 조정합니다. </li><li> 96 잘 접시의 우물에 무력화 콜라겐 솔루션 50 μl 추가 </li><li> 37 품어 ° C 콜라겐이 (90 분) 확정되기 전까지. </li><li> 얼음 methocel - 콜라겐 - 리간드 솔루션을 준비 : 콜라겐 솔루션의 각 리간드 피펫 한 부분. 콜라겐 TGF - β의 20 NG 당 ML을 추가합니다. 다른 레이어 위에 methocel 및 확산과 희석 후 TGF - β의 최종 농도 5 NG / vortexing에 의해 ML 믹스 것입니다. methocel - 솔루션의 한 부분을 추가합니다. vortexing하여 섞는다. </li></ol> 4. 회전 타원체 포함땡땡 <ol><li> 200 μl 피펫에, 팁 잘라 약 5mm가 200 μl 팁을 놓으십시오. 피펫 한 회전 타원체를 빨아 조심스럽게 빈 접시에 매체를 분배. 회전 타원체의 팁 내부 좋다면보세요. 회전 타원체는 흰색 점으로 표시됩니다. 플런저를 발표하지 않고, 100 μl 콜라겐 - methocel 혼합물을 빨아하고 콜라겐 - 코팅 잘 96에 콜라겐 혼합에있는 회전 타원체를 분배. 각각의 조건 12 spheroids에 대해이 작업을 수행합니다. </li><li> 콜라겐, 37 적어도 30 분 응고하자 ° C </li><li> 200 μl 팁과 공기 방울을 제거합니다. </li><li> 1.6 %의 혈청 및 DMSO에 녹아있는 저해제와 중간의 50 μl를 추가합니다. SB - 431542에 대한 재고 솔루션을 4 μl (10 ㎜) 중간 1 ML로 추가합니다. 확산 후 억제제의 최종 농도는 10 μm의 것입니다. TGF - β와 억제제는 실험의 과정을 계속합니다. 40X 배율을 사용하여 현미경 spheroids 사진을 가져가라. </li><li> 사 후TGF - β 및 / 또는 억제제와 자극의 8h는 40X 배율을 이용하여 현미경 spheroids의 사진을 찍을. </li><li> 이일 후에 침해 spheroids의 영역을 측정하고 시작 영역을 뺍니다. 의 영역을 측정 회전 타원체 : 어도비 포토샵 확장 (그림 2A)의 회전 타원체의 사진과 빠른 선택 도구 (그림 2B)을 선택 엽니다. &#39;플러스&#39;(+) 기호 (그림 2C)와 동그라미에 마우스 포인터가 변경됩니다. 회전 타원체 위에 커서를 드래그하는 동안 포토샵은 회전 타원체의 경계를 인식합니다. (-) 도구 모음에서 로그인 회전 타원체 외부 영역을 선택한 경우,이 지역은 Alt 키를 눌러 또는로 표시됩니다 부정적인 선택 도구를 사용하여 선택에서 제외시킬 수 있습니다. (-) &#39;마이너스&#39;로 동그라미에 커서 변경 서명하고 잘못 선택한 영역 위로 커서를 드래그하여,이 지역은 선택 (그림 2D)에서 제거됩니다. 필요한 여러 영역 쉬를 눌러 선택할 수있다면ift의 버튼을 클릭합니다. 더 정확하게 해결하려면 마우스 포인터의 크기는 도구 모음에서 브러쉬 직경 (그림 2E)을 변경하여 조정할 수 있습니다. 전체 회전 타원체를 선택하면, 선택 영역은 메뉴 막대에 분석 법안을 통해 또는 Ctrl + Shift + M (그림 2F)를 눌러 계산됩니다. 측정 기록 창에 파일 이름과 픽셀 선택 영역뿐만 아니라 다른 데이터 (그림 2G)를 보여주는 표시됩니다. 여러 선택이 측정하는 경우, 첫 번째 라인은 모든 영역의 합계를 보여줍니다. 개인 선택의 측정은 아래 라인에 제공됩니다. 모든 측정 데이터는 텍스트 파일에 수출 및 Excel에서 열 수 있습니다. </li></ol> 5. 대표 결과 : MCF10A1 (M1) 정상 유방 상피 세포와 회전 타원체 분석의 예를 들어 H - RAS는 MCF10AneoT (M2) 세포를 변환하고 M2 - 파생 MCF10CA1a (M4)는 그림 3에 있습니다. M1 세포없이 오직 약한 침략를 보여주자극하지만, 상당히 침공 TGF - β로 자극에 더. 대조적으로, M2는 자극의 추가없이 효율적으로 이미 침략 M1 - 유도체 RAS - 변환, 그리고 이것은 더 TGF - β 치료시 4-5 배 증가했다. M4 세포 모두와 TGF - β 이외없이 강한 침공을 보여주었다. 다음, 우리는 화학 억제제를 사용하여이 회전 타원체 모델에서 TGF - β - 유발 침공에 영향을 줄 수 있는지 여부를 조사했다. 이러한 목적을 위해 우리는 SB - 431542, ATP의 아날로그 및 TβRI의 키나제 활동의 선택적 억제제뿐만 아니라, activin 유형 IB 수용체 (ActRIB)와 activin 키나제와 같은 수용체 (하시죠) 7 12를 사용합니다. 예상했던대로, SB - 431542는 potently M1의 TGF - β - 유발 침공, 그 TGF - β - 유발 침략을 나타내는 M2와 M4 세포 (그림 4), TβRI - 키나제 저해 종속됩니다. 또한, spheroids의 기초 침공도 강하게 저해했다, 그 기저 침공을 제안하는 데 또한TGF - β에 늘어뜨린. <img alt="그림 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> 3D 회전 타원체 분석의 그림 1. 플로우 차트. 첫째, 세포는 U - 모양의 정지 플레이트에서 비 점착 성의 조건 하에서 도금입니다. 세포는 multcellular spheroids에 aggregrate과 콜라겐 매트릭스에 삽입하실 수 있습니다. 이 콜라겐 매트릭스에, 그들은 침략 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> 그림 2. 어도비 포토샵을 사용하여 spheroids의 지역의 부량가 확장. (A) 회전 타원체의 그림을 사용하여 wongly 포함된 영역의 회전 타원체 (D) 제거 영역을 선택 회전 타원체 위에 커서를 드래그 빠른 선택 도구 어도비 포토샵 Extendend (B) 선택 (C)에서 열립니다 더 정확하게 영역을 선택하는 빠른 선택 도구의 브러쉬 크기의 부정적인 빠른 선택 도구 (E) 조정 (F)가 선택측정 명령 (G) 측정 기록의 예를 기록합니다. <img alt="그림 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> 그림 3. 저절로 불후의 MCF10A1 (M1) (A)의 spheroids의 TGF - β - 유발 침공, MCF10AneoT (M2 (B) 및 전이성 파생 MCF10CA1a (M4) (C). M1, M2와 M4 spheroids는 임베디드 RAS - 변환 표시된로 콜라겐으로 48h에 대한 TGF - β의 5ng/ml로 치료했다. AC 대표 사진 포함의 하루 촬영하고 이틀 후. D 상대 공격이 2 일 마이너스 일 0에 지역 차이로 계량되었습니다. 결과 표현 같은 의미 ± SD 13에서 적응. <img alt="그림 4A" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="그림 4B" src="/files/ftp_upload/3337/3337fig4b.jpg" /> 그림 4. 자연적으로 흐르는 것과, 반사적으로 흐르는 및 TGF - β - 유도된 회전 타원체의 침공은 B를 저해합니다Y SB - 431542. M1, M2와 M4 spheroids은 콜라겐에 포함하고 5ng/ml TGF - β 및 표시 등 / 또는 10 μm의 SB - 431542로 치료했다. 이일 (A) 이후에 촬영한 대표 사진. 상대 침공은 2 일 마이너스 일 0 (B)에 지역 차이로 계량되었습니다. 결과는 말은 ± SD 13에서 적응으로 표현됩니다.

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> 30, 256-256 (2011).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-Activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-Activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> 10, 314-314 (2011).</li><li>Li, Q., Chow, A. B., Mattingly, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> J. Pharmacol. Exp. Ther. 332, 821-821 (2010).</li></ol>

관심 없음 충돌하지 않습니다.

우리는 MCF10A1 세포와 악성 유도체는 TGF - β - 의존적으로 콜라겐 매트릭스에 침공하는 회전 타원체 모델을 설립했습니다. 첫째, spheroids은 메틸 셀룰로오스의 면전에서 96 우물 원형 바닥 서스펜션 플레이트를 형성하고 있습니다. 물론, 또한 U - 모양의 폴리 - HEMA 코팅 플레이트는 이러한 목적을 위해 사용될 수 있습니다. 세포 메틸 셀룰로오스의 부재에 죽을 이후 메틸 셀룰로오스는 절대적으로,이 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> 메틸 셀룰로오스 </td><td> 시그마 - 올드 리치 </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> 고급 Biomatrix </td><td> 5005 - B </td><td></td></tr><tr><td> 산도 표시기 스트립 </td><td> 머크 </td><td> 9533 </td><td></td></tr><tr><td> 96 - 웰 정지 접시 </td><td> Greiner 바이오 - 원 </td><td> 650 185 </td><td></td></tr><tr><td> TC 접착 96 - 웰 접시 </td><td> Greiner 바이오 - 원 </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF - β는 초기 단계에서 종양 억제 역할을하지만, 나중에 무대 1,2에서 침략과 전이를 자극하여 유방암의 진행에서 역할을 반대했다. 또한, TGF - β가 자주 유방암에 overexpressed과 그 표현이 불쌍한 예후와 전이 3,4와 상호입니다. TGF - β 유도의 공격을 잘 이해하지하는 메커니즘. TGF - β는 TGF - β 타입 II (TβRII)와 유형 I (TβRI) 수용체를 통해 세포의 반응을 elicits. TGF - β - 유발 heteromeric 복잡한 형성되면, TβRII는 TβRI을 phosphorylates. 활성 TβRI는 phosphorylating 수용체 Smads (R - Smads), 즉 Smad2 및 Smad3하여 세포내 표준 신호 경로를 시작합니다. 이러한 활성 R - Smads는 핵에 축적하고 목표 유전자 5 전사를 조절 Smad4와 heteromeric 단지를 형성하고 있습니다. 이전에 D 이외에escribed Smad 경로, 여러 비 Smad 신호 경로의 활성화에 수용체 활성화 결과가 예를 들어 Mitogen는 단백질 키나제를 활성화하면 (MAPK) 경로 6. 유방암의 여러 단계에서 TGF - β의 역할을 연구하기 위해, 우리는 MCF10A 전지 시스템을 사용했다. 이 시스템은 자발적으로 남을 MCF10A1 (M1) 유방 상피 세포를 7로 구성되어, H - RAS는 생쥐 8 premalignant 병변을 생산 M1 - 파생 MCF10AneoT (M2), 그리고에서 설립되었습니다 M2 - 파생 MCF10CA1a (M4)를 변형 M2 xenografts 및 양식 폐 9 전이하는 능력을 가지고 고급 carcinomas. 이 MCF10A 시리즈는 다른 유전 배경에 의해 편견없는 강한 악의의 다른 성적 세포의 반응을 연구하기위한 가능성을 제공합니다. TGF - β - 유발 침략의 분석을 위해, 우리는 homotypic MCF10A 회전 타원체 셀 문화 embe를 생성시험 관내 (그림 1)에서 3D 콜라겐 매트릭스에 dded. 이러한 모델은 밀접하게 회전 타원체 10 내부에 괴사성도 외부와 정지 또는 세포에 대한 활성화 및 침입 세포의 결과로, 산소와 영양소의 그라데이션을 확립하여 생체내 인간의 종양과 유사. 회전 타원체 기반 assays는 monolayer 문화 11 이상의 약물 저항을 잘 요점을 되풀이하는 표시되었습니다. 이 MCF10 3D 모델 시스템은 우리가 강한 악의의 다른 단계에서 유방암 세포의 침투 특성에 TGF - β 신호의 영향을 조사 수있었습니다.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

양적 성장 인자 (TGF) - β - 유도 3 차원 콜라겐 젤의 침공을 변형 측정하는 분석이 설명되어 있습니다. 이 분석은 유방암 개발의 다른 단계를 나타내는 세포 라인의 MCF10A 시리즈의 활용합니다. 이 방법은 다른 세포 라인과 함께 사용하는 다른 가능성 activators 또는 침해의 억제제를 조사하기 위해 사용될 수 채택 수 있습니다.

TGF - β - 유발 침략을 측정하는 회전 타원체 분석

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

spheroid-assay-to-measure-tgf-induced-invasion

Research

JoVE Journal

Medicine

Spheroid Assay to Measure TGF-&#946;-induced Invasion

21.7K 조회수.  Leiden University Medical Centre. 양적 성장 인자 (TGF) - β - 유도 3 차원 콜라겐 젤의 침공을 변형 측정하는 분석이 설명되어 있습니다. 이 분석은 유방암 개발의 다른 단계를 나타내는 세포 라인의 MCF10A 시리즈의 활용합니다. 이 방법은 다른 세포 라인과 함께 사용하는 다른 가능성 activators 또는 침해의 억제제를 조사하기 위해 사용될 수 채택 수 있습니다.

비디오: Spheroid Assay to Measure TGF-&#946;-induced Invasion

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

암 세포에 의해 내피 세포 단층의 침략을 측정하는 실시간 전기 임피던스 기반 기술

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

연구

의학

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

축방향 및 근위 근육 톤을 측정하는 방법

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

진도 및 정밀 측정하는 치과, 새로운 방법의 정확도

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

실시간으로 중피 세포의 난소 암 회전 타원체의 부착과 침략의 평가

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

자궁 원격 측정은 마우스 수축 압력을 측정하는<em&gt; 생체</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

테스트를위한 병아리 심장 침략 분석 암 세포의 침윤과 잠재적 안티 침략 화합물의 활동

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

암 세포 회전 타원체 분석은 3D 설정에 침략을 평가하기 위해

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.

Here, we describe two measures of pulmonary function &#8211; barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

체적 Capnography와 기 Plethysmography 폐 구조-기능 관계를 측정 하

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...