우리는 임상 표본을 얻기 그녀의 귀중한 도움로나 랜쇼 (에딘버러 유방 단위)를 주셔서 감사합니다. 종양 소재의 이용은 로디언 연구 윤리위원회 (08/S1101/41)에 의해 승인되고 실험 암 의학 센터 프로그램 (에든버러)의 후원하에 얻은 것입니다. 이 작품은 스코틀랜드 기금위원회에 의해 지원됩니다. ADL은 미스 마가렛 버터 리키 신뢰 임상 사람이다.

1. 랫 뒷면에서 콜라겐을 추출 <ol><li> 70 % 에탄올에 냉동 신선한 쥐의 꼬리를 놓습니다. </li><li> 메스 노출 힘줄과 overlying 피부를 Deglove. </li><li> 멀리 꼬리와 소독을 70 % 에탄올의 장소에서 힘줄을 스트립. </li><li> 수집 힘줄과 초산 (250ml 0.5M 초산에 1g의 힘줄)로 전송을 나가는거야. 4 48hr에 대한 믹스 ° C. </li><li> 30 분 대한 1만g에 힘줄이 / 아세트산 혼합물을 원심 분리기와 펠렛을 삭제. </li><li> 뜨는 10 %의 동일한 부피 (W / V) NaCl을 추가하고 혼합 4에서 하룻밤 서있게 ° C. </li><li> G. 10,000에서 추가로 30 분 대한 콜라겐이 풍부한, 불용성 아래 레이어와 centrifugate를 수집 </li><li> 4 0.25M 초산에있는 콜라겐이 풍부한 재료 ° C를 Resuspend 두 번 매일 투석 버퍼를 변경, 3 일 동안 4 ° C에서 1:1000 초산에 대한 dialyse. </li><li> 또한 4 원심 분리 (2 시간에 대한 2만그램)과 저장하여 콜라겐 솔루션을 소독 ° C. </li><li> 1mg/ml의 재고 농도로 멸균 1:1000 초산의 추가로 필요한 콜라겐을 희석. </li></ol> 2. 3D 분석 작성 3D 분석은 세포 라인 분석을 기반으로, 이전에이 게시되었습니다. <ol><li> 다중 코어 biopsies는 침입 유방암에 대한 치료 수술 절제술시 환자 동의에서 수확하고 있습니다. </li><li> 눈으로, 1mm 3 조각으로 메스를 사용하여 코어를 나눕니다. 트림 및 macroscopically 별개의 지방을 삭제. </li><li> MEGM 완료 언론의 조각을 담가. 조직은 최대 1 시간까지이 상태로 저장할 수 있습니다. </li><li> 얼음 1mg/ml 랫 꼬리 콜라겐에있는 콜라겐 젤, 믹스를 생성하려면, 1:1000 필터링 초산, 3:5:1:1의 비율 10X DMEM/F12 및 0.22M NaOH 0.3의 최종 콜라겐 농도를 만들 수 MG / ML. </li><li> 37 인큐베이터에 24 잘 접시와 장소 개별 우물에 콜라겐 혼합의 1.2ml를 주사 ° C를 10 분 대한 겔화를 시작합니다. </li><li> 10 분 후 인큐베이터에서 24 잘 플레이트를 제거합니다. 겔화 콜라겐에 종양의 조각을 놓고 부드럽게 10μl 피펫 팁을 사용하여 젤의 중심에 그들을 밀어. 1 시간을위한 인큐베이터에 24 잘 번호판을 반환합니다. </li><li> ER + 종양 들어, 3.2x10 -10 M.의 농도 β - oestradiol와 MEGM 완전한 미디어를 보완 겔 assays는 1 시간에 대한 gelled 후에는 신중하게 각 우물에 보충 미디어 1.2ml 소개합니다. 검정 내에서 최종 β - oestradiol 농도는 유방 조직 3 발견 생리 에스 트로겐 수준을 요점을 되풀이하는 1.6x10 -10 M에 equilibriate 것입니다. </li><li> 따라서 각 젤 미디어 내에서 자유롭게 부동 수 있도록 circumferentially 각 젤 발표 각 우물의 안쪽 가장자리 주위에 10μl 피펫 팁을, 실행합니다. 인큐베이터에 24 잘 번호판을 반환합니다. </li><li> Exchange는 실험 종료까지 미디어가 두 번 주간 보충. 미디어를 제거하고 포르말린 정착액을 추가하여 실험을 종료할 수 있습니다. </li></ol> 3. 광학 프로젝션 Tomography 준비 어세이의 항체 스테 이닝 우리는 전체 샘플 얼룩의 4 이전에 게시된 방법을 채택했습니다. <ol><li> PBS로 30 분 대한 포르말린 고정 표본 씻으십시오. 각 단계에서 33% 66 %, 그리고 ≥ 15 100 % PBS는 0.05 % 트윈 20 (0.05 % PBST)을 포함하는으로 희석 메탄올에 stepwise 젤을 탈수. </li><li> DMSO : : 30 % H 2 autofluorescence를 끄지을 이용하려면에 대해 실온에서 2시 1분 3초의 비율 (15 % V / V H 2 O 2)에서 O 2 신선한 MeOH의 조직을 품어. </li><li> 메탄올에 30 분에 두 샘플을 씻으십시오. -80 ° C 조직의 깊은 부분에 항원을 보장하기 위해 실내 온도 (RT)로 적어도 1 시간마다 시간을위한 다섯 시간이 렌더링에 액세스할 수있는 샘플을 고정. </li><li> 희석제 (각 단계에서 33% 66 % 및 15에 대한 100 %)로 메탄올과 다시 0.05 %의 PBST로 조직을 Rehydrate. </li><li> 이용하려면에 대한 솔루션 (0.05 %의 PBST에서 혈청 10 %) 차단의 조직을 차단합니다. </li><li> 48hr 5 % DMSO를 포함 차단 솔루션 1.5ml에 희석 1:500의 농도에 방지 cytokeratin 토끼를 사용하여 상피 내용을 식별하는 기본 항체로 조직을 품어. </li><li> 30 분 각각에 대해 0.05 %의 PBST로 샘플 네 번이나 씻으십시오. </li><li> 48hr 5 %의 DMSO를 포함 1.5ml 차단 솔루션에 희석 형광등 보조 항체 (1:100 염소 방지 - 토끼 Alexa555)로 조직을 품어. </li><li> 30 분 각각 0.05 % PBST 네번로 다시 샘플을 씻으십시오. </li></ol> 4. 광학 프로젝션 Tomography 광학 프로젝션 tomography는 최대 15mm 5의 두께 형광 생물 학적 표본의 고해상도 3D 이미지를 생산하기 이전에 개발된 현미경 기법입니다. <ol><li> S 마운트tained 1퍼센트 낮은 용융 점 아가로 오스 겔 및 이용하려면에 대한 100 % 메탄올로 탈수. 48hr에 대한 밥 솔루션 (1시 2분 벤질 알코올, 벤질 벤조 산)의 표본을 해제하고 OPT 3001M 스캐너 (Bioptonics)을 사용하여 검사합니다. </li><li> 대상 및 autofluorescence 모두 Cy3 필터 세트 (자극 545nm/30 NM, 에미터 75분의 610 NM)와 각각 GFP1 필터 세트 (자극 425nm/40 NM, 에미터 LP475 NM)을 사용하여 캡처합니다. </li></ol> 5. 재건 및 3D의 부량 <ol><li> OPT 인수 각도 예측의 집합은 침공 분석을 통해 단면 슬라이스 일련의를 만드는 NRecon 소프트웨어 (SkyScan)를 사용하여 복원 수 있습니다. 이 소프트웨어에서 무료로 다운로드할 수 있습니다 <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Volocity 소프트웨어 (PerkinElmer)를 사용하여 복원 데이터를 Analyse. 무료 데모 소프트웨어는 다음 위치에서 다운로드할 수 있습니다 <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> cytokeratin 얼룩으로 상피 볼륨을 계량하기 위해서는 대상 채널의 배경 형광의 간섭을 줄이기 위해 최소 voxel의 강도 임계값 설정합니다. 우리는 측정 어플 리케이션에서 사용할 수있는 매개 변수 목록을 사용하여 9000 프에서 경험적으로이 임계값을 정의, 우리는 원래의 핵심 (autofluorescent 채널에 demarcated)과 연속성에서 상피의 볼륨을 계량. 이것은 우리가 다른 사이의 상피 가지의 볼륨을 계량 수 종양 샘플. </li></ol> 6. 대표 결과 : 분석의 개발 과정, 52 유방암의 총 조직 병리학의 다양한 표본이 사용되었습니다 생검. 이들 중 &lt;10 % (5 / 52) 적어도 하나의 가능한 공격 분석을 제공하지 못했습니다. 분석 실패도 위에 감염 세균이나 부족 종양 생검 재료에 의한했다. 따라서 분석의 응용 프로그램은 유방암의 특정 subtype에 제한되지 않습니다. 일반 assays (그림 1) 콜라겐 문화 20 일 후에 고정 및 이후 cytokeratin에 대한 얼룩. Volocity 소프트웨어는 원래의 핵심과 세포를 침해의 분리된 그룹과 지속적인 상피 대량을 모두 인식하고 개별적으로 각각의 볼륨을 정할 수 있습니다. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="그림 1" /> 고정 및 부량 다음 그림 1. 침공 assays. 검정 여러 유방암 subtypes에서 얻은 샘플을 지원합니다) ER + B) HER2 + 및 C) ER -.

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바느질과 JF은 Bioptonics OPT 스캐너의 상용화는 MRC 기술에 의해 고용됩니다. 기타 저자 관심에 대한 갈등을 선언하지 않습니다.

암 세포 라인의 행동을 연구하는 콜라겐 기반 assays 지금은 널리 6,7,8를 사용하고 있습니다. 그럼에도 불구하고, 이러한 assays는 종양과 환경의 전체 복잡에 반영하지 않았습니다. 본 연구에서는, 우리는 인간 유방암 자료는 유사한 방식으로, 전 생체내에서 사용할 수 있습니다 보여줍니다. 또한, OPT는 유방암의 3D 확장을 특징하는 유용한 도구입니다. 우리는이 기술의 주요 한계는 종양 자료 (단일 코어 생검은 4-6 assays에 충분합니다)의 이용되고있는 것으로 나타났습니다. 또 다른 제한 요인은 구조적 침략은 assays의 약 40 %에서 발생합니다. 이것을 극복하기 위해, 우리는 정기적으로 보이는 성장과 모든 assays하는 일 7.2 주변에 원하는 치료를 추가합니다. OPT의 사용은 또한 약물 치료로 인한 대량 변경, 전직의 생체내를 감지하는 미래의 확장 수 있습니다. 이 차례, 동물 모델의 독립보다 개별적인 암 치료를 용이하게 수 있습니다. 현재 tamoxifen 전직의 생체내에 ER + 종양의 반응을 탐구하고 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 완료 MEGM </td><td> Lonza </td><td> CC - 3150 </td></tr><tr><td> DMEM/F12 </td><td> 시그마 - 알드리치 </td><td> D8900 10X1L </td></tr><tr><td> β - oestradiol </td><td> 시그마 - 알드리치 </td><td> E8875 - 250MG </td></tr><tr><td> 토끼 방지 cytokeratin </td><td> Dako </td><td> Z0622 </td></tr><tr><td> 염소 안티 - 토끼 Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> 다른 모든 표준 시약은 시그마 - 알드리치 얻은되었습니다.

long-term culture of human breast cancer specimens and their analysis using optical projection tomography

유방암은 서양 세계에서 사망의 주요 원인이다. 이곳은 유방암의 확산은 먼저 로컬 나중에 distally, 환자 예후에 중요한 요인임을 설정됩니다. 유방암의 실험적 시스템은 일반적으로 기본 종양이나 흉막 effusions에서 파생된 세포 선에 의존하고 있습니다. 두 가지 주요 장애물이 연구를 저해 : (I) 유방 암 (특히 가난한 예후 luminal B 종양)의 하위 유형의 알려진 일부 현재 라인 컬렉션 내에 표시되지 않습니다; (2) 종양의 microenvironment의 영향은 일반적으로 고려되지 않습니다. 우리 모두는 하위 유형의 문화 일차 유방암 표본에 대한 기술을 보여줍니다. 이것은 종양의 작은 조각이 나는 부드러운 쿠션 쥐의 콜라겐에 포함된하는 입체 (3D) 문화 시스템을 사용하여 이루어진다. 2-3 주 내에, 종양 세포는 콜라겐으로 확산하고 인간 tumours1에서 관찰 비슷한 여러 구조를 형성하고 있습니다. 가능한 adipocytes는 원래 핵 내에 상피 세포와 섬유아 세포가 조직학에 분명했다. squamoid 형태와 악성 상피 세포는 주변의 콜라겐에 폐를 끼치고 시연했다. 핵 pleomorphism은 mitotic 인물과 apoptotic 기관과 함께,이 세포 내에서 분명했다. 우리는 문화의 종양 확산의 범위를 정할 수 있도록 광학 프로젝션 Tomography (OPT), 3D 이미징 기술을 고용했습니다. 우리는 종양 문화의 대량 볼륨을 측정하는 OPT를 사용하여 있고, 매개 변수는 정기적으로 약물 치료에 반응을 평가하는 유방암 환자의 네오 도움이되는 치료를하는 동안 측정. 여기, 우리는 하위 유형 편견없이 문화를 인간의 유방 종양 수있는 기회를 제시하고 그 전 생체내의 확산을 계량. 이 방법은 원래 종양의 약물 민감도를 수치 미래에 사용될 수 있습니다. 이것은 현재 사용되는 셀 라인보다 예측 모델을 제공할 수 있습니다.

Watch this Scientific Journal Video about Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography at JoVE.com

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

우리는 콜라겐 기반을 개발<em&gt; 체외에서</em모든 유방암 subtypes의 샘플에서 증식과 침략을 촉진&gt; 분석. 광학 프로젝션 Tomography, 입체 현미경 기술은 종양의 확장을 시각화하고 수치 활용했다. 이 분석은 개별 종양 샘플의 약물 반응을 계량하는 데 사용할 수 있습니다.

광학 프로젝션 Tomography를 사용하여 인간 유방암 표본과 그 분석의 장기 문화

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

long-term-culture-human-breast-cancer-specimens-their-analysis-using

Research

JoVE Journal

Biology

14.7K 조회수.  University of Edinburgh. 우리는 콜라겐 기반을 개발 체외에서 분석. 광학 프로젝션 Tomography, 입체 현미경 기술은 종양의 확장을 시각화하고 수치 활용했다. 이 분석은 개별 종양 샘플의 약물 반응을 계량하는 데 사용할 수 있습니다.

비디오: Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

연구

생물학

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

전체 심장 이미징에 대한 형광 광 프로젝션 Tomography에 대한 출생 정규화

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

기본 인간 난관 상피 세포의 전의 VIVO 문화

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

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 ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

엑손 캡처 및 대규모 병렬 염기 서열 종양 검체에서 체세포 유전 변경을 검출

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

다층 제브라 피쉬의 장기 라이트 시트 현미경에 장착

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

SCAP의 분석<em&gt; N</em&gt; 인간 세포의 -glycosylation 및 매매

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

성인 마우스및 쥐 심근세포의 격리, 트랜스페션 및 장기 문화

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...