We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

윤리 문 : 동물을 포함한 모든 연구 기관의 IACUC의 승인을 받아야합니다 (다음 데이터는, 프로토콜은 몬테 피오레 IACUC, 프로토콜 번호 11-6-240 및 # 13-7-100에 의해 승인되었다). 촬영시에는 여전히 그들을 유지하기위한 목적으로 지시 된 바와 같이 동물이 빛 마취를 제공,이 프로토콜에 대한 요구되는 고통스러운 절차는 없습니다. 사진 1. 취득 <ol><li> 인쇄 된 텍스트와 종이를 사용하여 겔 이미 저에 대한보기의 설정 포커스와 필드. 촬영 지역에 걸쳐 광원의 균일 성을 확인합니다. , 균일 성을 보장 반사 사진에 대한 광원 내장 된 젤 화상 카메라를 사용하는 데 도움이. 이 더 그레이 스케일 분석에 적합하지 않은 동물의 실루엣을 만드는 것처럼, transillumination 광원을 사용하지 마십시오. 참고 :이 약간 관심 (ROI)의 지역에 걸쳐 광학 평균을 제공한다 초점에서 마우스를 배치하고 의지합니다관심의 매우 작은 (즉, &lt;10 픽셀) 지역에 대한 계량 적 오류를 줄일 수 있습니다. 관심 영역 확대 핀트 조정이 마이너 의해 영향을받지 않으며 동물의 크기의 차이에 의해 영향을받을 것이다. </li><li> 케타민 (100 ㎎ / ㎖) / 자일 라진 (xylazine) (20 ㎎ / ㎖)를 사용하여 동물을 마취 (2 : 1)이 빠른 마취 효과와 신속한 복구를 제공하고 여러 동물을 촬영에 최적으로. 참고 : 동물이 스틸 사진을 허용하기에 충분 때 마취가 확인됩니다. 동물은 일반적으로 10 ~ 15 분 내에서이 빛 마취에서 회복으로, 수의사는 눈 연고의 사용을 권장하지 않았습니다. </li><li> (가까운 가능한 평행하는) 수직 정렬 젤 이미 저에 마취 동물을 놓습니다. <ol><li> 등의 사진의 경우, 확장 사지에 발생하기 쉬운 위치에 동물을 배치합니다. </li><li> 복부 사진의 경우, 동물이 옆으로 회전되지 않습니다주의하면서 앙와위에서 동물을 배치합니다. </li&gt; </ol></li><li> 촬영 지역의 그레이 스케일 표준을 놓습니다. </li><li> 닫기 액세스 도어. 중요 : 주변 광 노출의 변화를 소개 할 수 있습니다. </li><li> 인수의 선형 범위 내의 관심 영역을 배치 노광 F 스톱 세트 (F 스톱를 판독). 참고 : 이미지가 포화되는 경우 대부분의 시스템이 표시됩니다. </li><li> 사진을 가져 가라. </li><li> 변경 증가 또는 그러한 표준과 관심 영역 모두 취득 (기준 직선 범위에 있도록, 1 F 스톱을 감소시켜 선형 범위 내에서 관심 영역을 배치 다른 노출 설정으로 F는 스톱 F- ) 중지합니다. </li><li> 사진을 가져 가라. </li><li> 촬영이 완료되면, 온난화 테이블에 동물을 배치하고 그들이 흉골 드러 누움을 유지 할 수있을 때까지 모니터링 할 수 있습니다. 새장에 동물을 돌려줍니다. 한 번에 모든 동물이 완전히 회복 된 동식물 사육장에 동물의 그룹을 돌려줍니다. </li></ol> 광 흡수 2. 정량 &lt;/ P&gt; <ol><li> 동물의 이미지에 대한이자의 마크 영역은 젤 이미 저를위한 소프트웨어를 제공하는 사용. <ol><li> 전체 동물의 등쪽 뷰 가능한 횡만큼 연장되는 하부 사지 상지로부터 연장되는 직사각형 또는 타원형 이미지를 사용하도록도 1a에 도시 된 바와 같이 상자의 일부 동물의이면 넘어 연장하지 않는다. 참고 : 하나는 또한 프리 핸드 드로잉 도구를 사용하여 관심 영역을 표시 할 수 있습니다. </li><li> 골반 영역을 덮는 온 및도 1b에 도시 된 바와 같이, 가슴 부분을 커버 하나 동물 전체 뷰를 들어 복부,이 사각형을 사용한다. </li><li> 적절한 관심, 즉, 약물 투여의 위치, 마크의 작은 지역. </li></ol></li><li> 그레이 스케일 흡수 표준에 대한 관심의 표시 영역 (들). </li><li> 관심 표시된 영역 각각에서 기록 흡수. </li></ol> 3. 분석 흡수 레벨 obtaine관심 지역에 대한 D는 사진의 비교에 대한 배경 표준을 정규화해야 할 수도 있습니다. 흡수에 노출 관계는 로그 - 로그 (즉, 관계가 로그 (노광 간의 선형 임) 및 로그 (흡수))이다. 이 관계를 사용하여, 관심 영역의 흡수는 흡수도가 상이한 시점 (연구 프로토콜 내에서, 즉, 일련의 측정)에서 찍은 포함한 다른 사진 사이에 직접적으로 비교 될 수 있도록 표준 배경 값으로 정규화 될 수있다. 이러한 정정을 수행하기위한 절차는 선택적 단계 3.1 및 3.2에 설명된다. <ol><li> 그레이 스케일 흡수 표준에서 얻은 값을 사용하여 로그 (흡수) 대 로그 (노출)의 (선택 사항) 플롯 곡선. </li><li> 다음 (선택 사항) 각 사진의 표준 편차를 조정합니다 : <ol><li> 의 결정으로 (참조 및 F 스톱 (단계 1.6에서 결정) 읽기 위해 F-중지를 선택) 1.8 TEP. </li><li> 독서 F-정류장에서 모든 사진에 표준의 평균 흡수를 계산합니다. 이 평균은 참조 표준 값 (RSV)입니다. </li><li> (델타 ROI-1, 델타 ROI-2 ... ..delta-ROI-X) 표준 (델타-S)에 대한 각각의 정의 ROI에 대한 각각의 사진에 읽기 및 참조 F-정지 설정 사이의 흡수의 차이를 계산 </li><li> 다음과 같이 각각의 투자 수익 (ROI)에 대한 수정 흡수를 계산 : 수정 흡수 (ROI-X) = 흡수 (ROI-X)를 읽고 F-정류장에서 + (RSV -에서 표준의 흡수 읽는 F-정지) * (델타 ROI-X / 델타-S) </li></ol></li><li> 실험 데이터를 컴파일하고 연속 변수에 대한 표준 통계 기법을 사용하여 데이터 분석을 수행 (즉, ANOVA, T-시험). </li></ol>

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Authors have nothing to disclose.

이 보고서에서, 상세한 설명은 설치류 탈모 정​​량화 새로운 기술이 제공된다. 이 기술은 대부분의 실험실에서 쉽게 사용할 이미지 수집 및 분석, 장비 젤 이미 저를 사용합니다. 측정 기법에서 작은 변화 (도 4-5)에 강인하도록 도시되었으며, 치료 모델에서 시각적 탈모 (도 6)의 정도에 잘 상관된다. 이러한 기술들은 성공적 탈모증 (17)의 평가 실험 치료에 사용?...

탈모증 (탈모)는 여러 원인으로 심리적으로 감정적으로 고통 이벤트가 될 수 있습니다. 남성 패턴 대머리는 35 세 1로 남성의 약 3 분의 2에 영향을 미치는 탈모의 가장 흔한 원인이다. 탈모의 유사한 패턴은 다낭성 난소 증후군을 가진 여성에서 관찰 할 수있다. 이러한 질환의 모두에서, 탈모 안드로겐 매개된다. 탈모증은 인구 2의 1.7 %에 영향을 미치는자가 면역 질환, 원형 탈모증으로 발생할 수 있습니다. 탈모는 화학 요법과 같은 세 응급 치료의 부작용으로 발생할 수있다. 화학 요법 환자의 높은 비율 (65~85%)는 탈모 4,5 어느 정도 경험한다. 탈모의 심리적 영향은 잘 설정 화학 요법으로 연구되어왔다. 화학 요법에 의한 탈모는 불안, 우울증, 부정적인 신체 이미지 저하 자부심과 6,7 복지의 축소 된 의미에서 발생할 수 있습니다. 높은 체육여성 암 환자의 rcentage (47-58%)는 화학 요법의 가장 충격적인 양상으로 탈모를 고려, 8 % 감소 치료까지 탈모 4,6의 두려움. 탈모 8,9 심리적 심지어 의료 결과를 줄이는 치료를 지원하는 안드로겐 성 탈모증의 증거가있다. 마찬가지로, 원형 탈모증은 심리적 영향이 심한 것으로보고되었으며, 탈모의 고르지 자연 탈모의 원인은 대부분의 다른보다 더 불쾌한 화장품 결과로 이어질 수있다. 가벼운 항 안드로겐 효과 (예, 스피로 노 락톤)와 약물이 탈모에 대한 치료와 같은 제한적인 성공에 사용 된 반면, 탈모의 첫 번째 효과적인 약물은 미녹시딜 (10)이었다. 이러한 항 고혈압은 모발 성장을 야기 관찰 부작​​용을 갖고, 이제 많은 형태의 탈모증 용 국소 요법으로서 사용된다. 그러나 반응은 일부 과목 만 느린 보여주는 종종 불완전실제 재성장 (10)보다는 탈모 보내고. 피나스테라이드 부분 전신 안드로겐 봉쇄의 비용 안드로겐 탈모증의 개선의 결과로, 디 하이드로 테스토스테론을 차단 변환 II 5α 환원 효소를 입력 할 수있는 경쟁력 길항제이다. 장기 (10 년) 치료와 응답의 비율은 약 50 % (11)이다. 전반적으로,이 분야에서 상당한 연구에도 불구하고, 여전히 탈모에 대한 적절한 치료가 없다. 수십 년 동안 과학자와 임상 임상 시험에서 두피 모발 성장을 측정하는 방법을 살펴 보았다. 탈모증을 치료 약물의 개발로, 모발 성장 및 치료에 특히 응답을 측정, 경제적이고 신뢰성있는 최소 침습 수단 클 필요가 있었다. 탈모 질환 환자의 머리 밀도의 정확한 정량에 대한 이미지 분석 기술은 techniqu의 다양한 사용하여 과거에 일관되고 유효한 결과를 산출디지털 이미지 (12)의 분석, 개별 머리카락의 이미지 분석과 피부 (13) 병변 및 정의 두피 영역 (14)의 머리 질량을 정량화 현미경 스캐닝을 포함하여, ES. 위의 방법은 머리가 임상 시험에서 개입을 성장 촉진 효과의 개선 된 평가를 제공하고 불행히도, 이러한 방법은 전임상 연구에서 설치류 연구에 적용되지 않았다. 우리의 목표는 연구자가보다 정확하게 평가하고 탈모의 다양한 형태에 대한 새로운 치료 방법을 비교 할 수 마우스에서 탈모를 정량화 할 수있는 일관되고 신뢰할 수있는 방법을 개발하는 것입니다. 우리는 갈색 또는 검은 색 머리를 가진 생쥐의 머리 밀도의 신속하고 신뢰할 수있는 정량을 수 있습니다 대부분의 실험실에서 쉽게 사용할 장비를 사용하는 방법을 개발했다. 이 방법은 waxin에서 화학 요법에 의한 탈모증, 원형 탈모증, 탈모증의 마우스 모델에서 테스트되었습니다g. 자세한 프로토콜 검증 C57BL / 6의 데이터와 C3H / 마우스의 HEJ 균주를 포함하여이 측정을 수행하기 위해 제공됩니다. 이 기술은 모간의 안료에서 광 흡수를 검출에 의존 같이, 흰색 알비노 마우스 또는 마우스에서 모발 성장을 검출하는데 사용될 수 없다.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">KODAK Gel Logic 100 Imaging System&#160;&#160;</td>
			<td style="width:121px;">Eastman Kodak Company, Rochester, NY, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">The Kodak/Tiffen Q-13 Gray Scale</td>
			<td style="width:121px;">Imatest, http://www.imatest.com/docs/q13/</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Greyscale standard</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J Mice</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C3H/HeJ engrafted mouse</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
	</tbody>
</table>

a new technique for quantitative analysis of hair loss in mice using grayscale analysis

탈모증 남성형 탈모, 다낭성 난소 증후군, 및 원형 탈모증 등 다양한 조건에서 발생할 수있는 탈모의 일반적인 형태이다. 탈모증은 암 환자의 화학 요법의 부작용으로 발생할 수있다. 이 연구에서 우리의 목표는 연구자가 정확하게 평가하고 탈모의 이러한 다양한 형태의 새로운 치료 방법을 비교 할 수 마우스에서 탈모를 정량화 할 수있는 일관되고 신뢰할 수있는 방법을 개발하는 것이 었습니다. 방법 마우스 검정 (또는 회색) 모발의 양에 비례하여 발생 거친 광 흡수를 측정 마우스의 이미지를 획득하고 처리하는 표준 겔 이미 저를 이용한다. 이러한 방식으로 정량화 된 데이터는 표준 통계 기법 (즉, ANOVA, T 시험)을 사용하여 분석 될 수있다. 이 방법은 왁싱에서 화학 요법에 의한 탈모증, 원형 탈모증과 탈모의 마우스 모델에서 테스트되었습니다. 이 보고서에서 상세한 프로토콜 대가입니다ented 검증 C57BL / 6의 데이터와 C3H / 마우스의 HEJ 균주를 포함하여 이러한 측정을 수행합니다. 이 새로운 기술은 애플리케이션의 상대적인 단순성, 대부분의 연구 실험실에서 쉽게 사용할 장비에 의존하고 대물인가 주관적인 평가보다 더 강력하다 정량적 평가를 포함하여 다수의 이점을 제공한다. 쥐의 모발 성장의 정량화 개선 탈모증 모델의 연구를 개선하고 전임상 연구에서 유망한 새로운 치료법의 평가를 더 용이하게 할 것입니다.

이 기술은 C3H / 마우스 개체로 이식 HEJ 16 원형 탈모증 탈모증 마우스 모델을 이용하여 검증 하였다. 이러한 동물은 시간에 따라 서서히 진행 글로벌 탈모를 개발한다. 그러나, 탈모 패치에서 발생, 상당한 변동성을 도입하고 질적 평가를 방해 한 마우스에서 다음에 따라 달라질 수 있습니다. 이 모델은 유효성의 평가로서 마우스의 상이한 위치에서 측정 한 광학 밀도 사이의 상관 관계를 ?...

Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

그레이 스케일 분석을 사용하여 마우스의 탈모의 정량 분석​​을위한 새로운 기술

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

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8.9K 조회수.  Children's Hospital at Montefiore. Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

비디오: A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

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 ...

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생물학

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

라이브 셀 이미징 및 배아 상피 세포의 정량 분석 Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

분열 효모의 양적 라이브 세포 형광 - 현미경 분석

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

시간 경과 비디오 현미경을 이용한 세포의 무작위 마이 그 레이션의 정량 분석

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

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

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

양적 휘트니스 분석 워크플로우

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

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

헤어 재생시 상피 - 중간 엽 시그널링의 빠른 유전 분석

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

고급 3D 형광 현미경을 사용하여 자식 작용의 정량 분석

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

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 ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

multiprotein의 단지의 비교 분석을위한 새로운 접근 방식을 기반으로<sup&gt; 15</sup&gt; N 대사 라벨링 및 정량 질량 분석

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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

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

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

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

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

그레이 스케일 분석을 사용하여 마우스의 탈모의 정량 분석​​을위한 새로운 기술

그레이 스케일 분석을 사용하여 마우스의 탈모의 정량 분석을위한 새로운 기술