저자는 인정 하는 없다.

1. 세포 배양 수집을 모니터링
<ol><li>DMEM + 글루타민 (예, GlutaMAX) 헬러 세포 성장 매체 10% (v/v) 열 비활성화 태아 송아지 혈 청, 1% 페니실린과 스 보완.</li>
	<li>6 잘 유리 하단 문화 접시 fibronectin (25 µ g/mL) 1 h 코트. 다음에 잘 당 2 × 104 셀 씨.</li>
	<li>인수, 동안 매체를 3 일 마다 변경 합니다.</li>
	<li>시간 경과 수집에 대 한 비디오 렌즈 무료 현미경 (상용)을 사용 합니다. 참고: 이것은 근거한 전산 렌즈 무료 이미징 기술에 Ozcan 그 외 여러분 에 의해 설명 된 대로 16 는 37 ° C12,15의 제어 온도에 인큐베이터에서 지속적인 모니터링을 수행 하도록 수정 되었습니다. 그것은 1.67 μ m의 픽셀 피치와 6.4 x 4.6 m m2의 이미징 영역 보완 금속 산화물 반도체 (CMOS) 이미지 센서를 갖추고 있습니다. 여러 파장 조명 장치에 의해 멀티 칩 빛 발광 다이오드 (Led), 빨강, 녹색 및 파랑 조명 제공 제공 됩니다. 파장은 중심, 636, 521, 452 nm 각각 25, 45, 25의 스펙트럼 대역폭으로 nm 각각. 빨강-녹색-파랑 (RGB) Led 세포에서 약 5 cm의 거리에서 150 µ m pinhole 위에 있습니다. 빛 전원 LED 주변 측정은 각 파장에 10 µW 낮은 이며 조명 시간 인수 당만 1 초. 따라서, 셀 문화는 렌즈 무료 비디오 현미경 관찰 사진 독성 문제가 예상 된다.</li>
	<li>셀 문화 컨테이너 CMOS 센서 (그림 1)과 접촉을 넣어. 컨테이너를 사용 하 여 렌즈 무료 취득의 품질에 대 한 중요 한 맨 유리 coverslip로. 플라스틱 용기를 사용할 수 있습니다 하지만 렌즈 무료 취득 이러한 컨테이너의 광학 품질 때문에 타락 될 것 이다.</li>
	<li>제어 비디오 렌즈 무료 현미경 수집 (상용) 소프트웨어와 함께 시간 경과 수집 및 홀로그램 재구성을 수행 합니다. 소프트웨어 인터페이스에서 사용자가 입력 해야 하는 매개 변수는 프레임 속도 실험, 세포 배양, 즉 부착 셀 또는 부동 셀의 종류의 기간.</li>
	<li>수집 소프트웨어의 입력된 매개 변수를 설정 합니다. 1 수집 10 분 간격으로 설정 하 고 '부착' 셀 문화 유형을 설정 프레임 속도 설정 합니다. 참고: 한 인수 10 분 마다의 프레임 속도 좋은 타협으로 안정적인 셀 추적 분석 결과 데이터 집합의 크기는 여전히 합리적인 보장. 빠른 프레임 속도이 현미경 렌즈 무료 설치와 함께 달성 한 인수 5 분 마다입니다. 과도 한 난방 문화에 있는 세포의 생존 능력에 영향을 미칠 수 있는 CMOS 센서의 결과 프레임 속도 더 증가.</li>
	<li>시간 경과 수집을 시작 하 고 홀로그램 재구성 알고리즘 (상용) 세포 배양의 위상 이미지를 렌즈 무료 수집을 자동으로 처리 됩니다. 홀로그램 재구성 알고리즘 기반으로 다중 파장 위상 검색 알고리즘17 하며 RGB 재건된 단계 이미지의 범위에 [-π, π +] 29.4 m m2의 넓은 시야를 통해 각 단일 셀에 대 한. 밝히는 얇은 샘플 렌즈 무료 인라인 홀로 그래피의 원리는 (z = 0) (진폭 1 정규화 후)의 평면 파로 짧은 거리 z에 후속 회절 강도 이미지 CMOS 센서에 감지 하 고 = Z, 후 일반적으로 1 m m는 샘플입니다. 우리의 설정에서 조명 순차적으로 전환 3 파장 (λ1= 0.450 µ m 이다, λ20.540 µ m 이다, λ3= = 0.647 µ m) 동일한 샘플의 3 개의 회절 패턴 측정 하.</li>
</ol>2. 세포 문화 데이터 분석
<ol><li>셀 추적용 Trackmate 알고리즘, 단일 입자18의 자동화 된 추적에 대 한 오픈 소스 피지 플러그인을 사용 합니다. 에 먼저, 피지 (부하 이미지 시퀀스 명령)에 전체 시간 경과 수집 로드. 일반적으로 400 RGB 프레임 29.7 Mb의 기능, 데이터 집합의 큰 크기 때문에 빠릅니다 8 비트 포맷에 그들을 로드. 다음 Trackmate 피지 플러그인 즉 셀 감지 단계, 셀 추적 단계 및 셀 탐지 및 계산된 트랙에 적용 되는 여러 필터 셀 추적 알고리즘의 여러 단계를 통해 사용자를 안내 합니다. 모든 단계에서 알고리즘을 구성 하 고 결과 표시 즉시를, 필요한 경우, 하나 쉽게 이동할 수 있습니다와 설정을 재조정. Trackmate 인터페이스의 다양 한 메뉴는 HeLa 세포 실험에 사용 된 실제 설정 함께 그림 2 에 표시 됩니다.</li>
	<li>( 그림 2참조) 다음과 같은 수집 소프트웨어의 입력된 매개 변수를 설정 합니다. 15 픽셀, 15 픽셀 간격 닫기 최대 거리 및 3.5 트랙에 관광 명소의 수를 예상된 blob 직경 15 픽셀, 0.25 검출기 임계값, 연결 최대 거리를 설정 합니다. 참고: 이러한 설정은 분명 다릅니다 한 실험에서 특히 '예상된 blob 직경' (1.67 μ m의 픽셀에 주어진 여기) 셀 크기에 해당 하는 다른. 같은 발언 '연결 최대 거리' 최대 거리에 대 한 계정 (1.67 μ m의 픽셀에 주어진 여기)의 연속 프레임 두 개 내 셀 여행에 적용 됩니다. 최적의 값 셀 운동에 뿐만 아니라 세포 밀도에 따라 것입니다.</li>
	<li>셀 추적 과정의 끝에, 세 개의 텍스트 파일의 형태로 결과 생성 ('분석' 버튼을, 그림 2참조). 인수, 그들의 프레임 번호 및 트랙 번호에 그들의 각각 (x, y) 위치와 검색 된 모든 셀을 나열 하는 테이블 '트랙 통계의 관광 명소' 라는 가장 유용한 파일에 의하여 이루어져 있다. 이 데이터를 기준으로 셀 세분화를 수행 하 고 전용된 알고리즘 (보충 코드 파일 참조)를 사용 하 여 세포 분열을 감지. 두 개의 다른 파일 이름이 '트랙 통계에서 링크'와 '통계를 추적 하 는'와 트랙, 트랙 기간 예를 들면 , 감지 된 간격의 수를 취급 하는 모든 결과 포함, 초기 프레임, 등을 추적.</li>
	<li>셀 분할 알고리즘을 사용 하 여 재건된 단계 이미지에서 셀 형태를 설명 하는 몇 가지 통계를 자동으로 추출 (참조 보충 코드 파일). 이러한 메트릭은 셀 표면적 (S), 세포 주 축 길이 (L), 셀 부 축 길이 (l)와 셀 가로 세로 비율 (L/l)입니다. 위상 현미경 렌즈 무료에서 발견 한 듯이15표본 층의 두께 밀도에 비례 이다.</li>
	<li>세포의 형태학 적 기능 외에 재건된 단계13,,1920 에서 양적 셀 건조 질량 (CDM) 측정을 추출 <img alt="Equation 1" src="/files/ftp_upload/56580/56580eq1v3.jpg">식 (1) φ(x,y)는 재건된 단계 교대19, λ는 파장 및 α = 1.8 x 10-4 m3/k g 건조 질량1, 굴절률의 변화에 관련 된 특정 굴절률 19.</li>
	<li>문화 매체의 굴절률 셀 사이의 차이의 추정을 사용 하 여 셀 간격을 평가 합니다. 셀 두께 h(x,y)와 측정 된 위상 변화 사이의 관계는20에 의해 주어진 다: <img alt="Equation 2" src="/files/ftp_upload/56580/56580eq2v3.jpg">식 (2.1) <img alt="Equation 3" src="/files/ftp_upload/56580/56580eq3v3.jpg">식 (2.2) Δn(x,y) = n셀 (x,y)- n매체 (x,y), 셀 및 문화 매체의 굴절률 차이. 다음, 가정 Δ의 상수 값n 0.025 =, HeLa 세포 핵에서 측정 하는 굴절률에서 추정 되는 (n = 1.35511) 인산 염 버퍼 식 염 수 (PBS) 문화 미디어의 (n = 1.33). 셀 간격 값은 가정에 기반으로 나타내는, 상대 유사 의미 하 고 나누어 셀을 검출 악용 될 수 있습니다.</li>
	<li>전용된 알고리즘을 사용 하 여 측정 두께 8 µ m 보다 큰 세포가 세포 분열의 발생을 감지 하 여 다음 세포 분열을 탐지 하기 위해 검색 된 셀 사단에 관련 된 셀 트랙을 추출 (참조 보충 코드 파일) 먼저 식별 됩니다, 그리고 확인 여부는 새 셀에 밀접 하 게 나타납니다 공간 시간. 이 방법에서는, 세포 분열의 감지 두 강력한 조건에 의존합니다. 세포 분열 탐지를 위한 더 정교한 방법과 대안 lensfree 시간 경과 인수21,,2223에 적용할 수 있습니다.</li>
</ol>

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M., Schicktanz, S., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+validation+algorithm+allows+for+automated+cell+tracking+and+the+extraction+of+biologically+meaningful+parameters.">A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters.</a> PLoS One. 6 (11), e27315 (2011).</li><li>Al-Kofahi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+cell+lineage+construction:+A+rapid+method+to+analyze+clonal+development+established+with+murine+neural+progenitor+cells.">Automated cell lineage construction: A rapid method to analyze clonal development established with murine neural progenitor cells.</a> Cell Cycle. 5 (3), 327-335 (2006).</li><li>Meijering, E., Dzyubachyk, O., Smal, I., van Cappellen, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+in+cell+and+developmental+biology.">Tracking in cell and developmental biology.</a> Semin Cell Dev Biol. 20 (8), 894-902 (2009).</li><li>Posakony, J. W., England, J. M., Attardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+growth+and+division+during+the+cell+cycle+in+HeLa+cells.">Mitochondrial growth and division during the cell cycle in HeLa cells.</a> J Cell Biol. 74 (2), 468-491 (1977).</li><li>Zocchi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD38+Increases+Intracellular+Calcium+Concentration+and+Reduces+Doubling+Time+in+HeLa+and+3T3+Cells.">Expression of CD38 Increases Intracellular Calcium Concentration and Reduces Doubling Time in HeLa and 3T3 Cells.</a> J Biol Chem. 273 (14), 8017-8024 (1979).</li><li>Reitzer, L. J., Wice, B. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Antisense+RNA+against+Initiation+Factor+eIF-4E+mRNA+in+HeLa+Cells+Results+in+Lengthened+Cell+Division+Times,+Diminished+Translation+Rates,+and+Reduced+Levels+of+Both+eIF-4E+and+the+p220+Component+of+eIF-4F.">Expression of Antisense RNA against Initiation Factor eIF-4E mRNA in HeLa Cells Results in Lengthened Cell Division Times, Diminished Translation Rates, and Reduced Levels of Both eIF-4E and the p220 Component of eIF-4F.</a> Mol Cell Biol. 11 (11), 5435-5445 (1991).</li><li>Kumei, Y., Nakajima, T., Sato, A., Kamata, N., Enomoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+G1+phase+duration+and+enhancement+of+c-myc+gene+expression+in+HeLa+cells+at+hypergravity.">Reduction of G1 phase duration and enhancement of c-myc gene expression in HeLa cells at hypergravity.</a> J Cell Sci. 93 (2), 221-226 (1989).</li><li>Ginzberg, M. B., Kafri, R., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+being+the+right+(cell)+size.">On being the right (cell) size.</a> Science. 348 (6236), 1245075 (2015).</li><li>Mathieu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+lens-free+imaging+of+cell+migration+in+diverse+physical+microenvironments.">Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.</a> Lab Chip. 16 (17), 3304-3316 (2016).</li></ol>

저자는 공개 없다.

이 문서에서 우리는 그 렌즈 무료 비디오 현미경 세포의 수천의 활동을 캡처 인큐베이터 안에 사용할 수 있습니다 보여줍니다. 전반적인 방법론을 설명 하기 위해 우리는 어떻게 문화에 HeLa 세포의 2.7 일 경과 수집 표준 셀 추적 알고리즘으로 분석 될 수 있다 설명 했다. 2.2 x 106 셀 측정 및 10,584 세포 주기 트랙을 갖춘 데이터 집합입니다. 인수는 비교적 큰 셀 셀 거리 Hela 세포의 문화에 수행 (셀 밀도 < 500 세포/mm2) 세포 형태학 디스크 접근 될 수 있다. 이 두 기능은 같은 큰 데이터 집합을 수집 가능 하 게 하는 자동 셀 추적 분석의 응용 프로그램을 촉진 한다. 다른 셀 라인 작은 셀 셀 거리 또는 더 복잡 한 형태학 추적 하기 더 어려울 것 이다 고 후속 데이터 집합 더 작을 것 이다.
우리는이 이렇게 많은 연구, 예를 들어 세포 증식과 세포 크기 연구에 대 한 흥미로운 믿습니다. 후자는 기본적인 생물학 연구의 중요 한 영역 및 많은 질문이 대답 없 남아 있다 멋지게 Ginzberg 외. 여 리뷰에서 설명한 29.이 작품에서 제안 된 프로토콜 제공 따라서 중요 한 통계 수치를 측정 하는 수단 셀 확산 연구, 즉 세포 주기 기간에 필요한 셀 건조 질량, 주기와 셀의 시작과 끝 사이 셀 건조 대량 비율 마른 대량 성장 속도입니다. 세포 증식의 연구 외에도이 방법은 생산할 수 있는 많은 트랙, 몇 시간을 지속 한 이후 세포 이동의 연구에 대 한 관심의 있을 것입니다. 현재 프로토콜 10 h 동안 셀 트랙의 수천의 인수를 처음으로 시연 하 여 첨단14,30 넘어 갑니다.
결론적으로, 우리는 사용 하기 쉬운 렌즈 무료 기술 문화의 몇 일 동안 레이블 없는 세포의 수천을 동시에 추적할 수 있게 개발 했습니다.

여러 가지 세포 주기 전체에 걸쳐 경작된 한 포유류 세포를 모니터링 하 고 정확 하 게 측정 셀 크기와 셀 건조 질량은 어려운 작업. 여러 레이블 없는 광학 기술이 작업1,2를 수행할 수 있습니다: 위상 이동 간섭계3, 디지털 홀로그램 현미경 (DHM)4,,56, 7, quadriwave 측면 전단 간섭계8,9 그리고 양적 단계 단층 촬영10,11. 이러한 방법은 포유류 세포의 세포 주기의 이해에 많은 새로운 통찰력을 끌고있다. 그러나 그들은 거의 알고리즘을 추적 하는 자동 셀과 결합 된 측정 될 때 그들의 처리량 제한 유지 세포 대량 궤도1 (N < 각각3,,45 에서 20 , 6). 따라서 소설 광 방법 큰 통계와 셀 대용량 궤적을 측정 필요 (N > 1000).
이 문서에 동시에 인큐베이터, 내부 직접 세포의 이미지 다음 개별 세포 주기 트랙의 수천에 따라 단일 셀 통계를 계량을 렌즈 무료 비디오 현미경의 기능을 설명 합니다. 현미경 렌즈 무료 양적 위상 이미징 기술을 매우 큰 시야에 밀도가 포장된 셀의 위상 이미지의 인수를 허용 하는 (일반적으로 여러 가지 수만 m m2, 여기 29.4 m m2)12,13 ,,1415. 단일 셀 수준에서 몇 가지 통계 결정 됩니다, 예를 들어, 셀 지역, 건조 질량, 셀 간격, 셀 주요 축 길이 셀 및 가로 세로 비율12,15, 각 이미지에서 셀. 그런 다음 셀 추적 알고리즘을 적용 하 여 이러한 기능 그릴 수 있습니다 모든 단일 셀에 대 한 실험 시간14,15의 기능으로. 또한, 셀 트랙에 세포 분열의 발생을 감지 하 여 그것은 초기 세포 (세포 분열) 후, (세포 분열) 직전 최종 셀 건조 질량 질량과 셀 건조와 같은 다른 중요 한 정보를 추출할 수 기간, 즉, 2 개의 연속적인 사단15시간 주기. 모든이 측정은 매우 좋은 통계 계산 될 수 있다 (N > 1000) 이후 큰 시야 일반적으로 200 ~ 10000 셀 단일 렌즈 무료 획득의 분석 허용.
렌즈 무료 비디오 현미경 검사 법에 따라이 방법론을 설명 하기 위하여 감시 하 고 2.7 일 HeLa 세포 배양을 계량 프로토콜을 설명 합니다. 데이터 분석은 다중 파장 홀로그램 재건, 셀 추적, 셀 분할 및 세포 분열 알고리즘에 따라 4 단계 프로세스입니다. 여기 공간 해상도이 렌즈 무료 비디오 현미경 설치 얻은 비교적 빠른 프레임 속도 (한 인수 10 분 마다) 표준 셀 추적 알고리즘와 호환 되는지 표시 됩니다. 이 데이터 집합의 전체 분석 완전 한 세포 주기에 10,584 셀 트랙의 측정에서 발생합니다.
요약, 렌즈 무료 비디오 현미경은 자동으로 레이블이, 동기화, 수천 및 실험; 당 수정 되지 않은 셀을 모니터링 하는 강력한 도구 여러 가지 세포 주기를 통해 추적 되 고 각 셀입니다. 우리의 측정은 따라서 세포의 큰 인구에 간 세포 가변성을 더 중요 하 게, 하지만 몇 가지 셀 매개 변수 평균 값을 제공합니다.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Cytonote lens-free video microscope&#160;</td>
			<td style="width:208px;">Iprasense</td>
			<td style="width:156px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horus acquisition software</td>
			<td>Iprasense</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well glass bottom culture plates</td>
			<td>MatTek corporation</td>
			<td>Part No: P06G-0-14-F&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM + GlutaMAX medium&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat-inactivated fetal calf serum&#160;</td>
			<td>Eurobio</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin and streptomycin&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin&#160;</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matlab, image processing toolbox&#160;</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture

여기, 우리가 보여 그 렌즈 무료 비디오 현미경을 동시에 인큐베이터 안에 직접 세포의 수천의 활동을 캡처 수 있습니다 하 고 모니터링 하 고 여러 세포 주기에 따라 단일 셀 계량 가능 하다. 우리는 모니터 2.7 일 HeLa 세포 배양을 계량 하는 데 사용 하는 전체 프로토콜을 설명 합니다. 첫째, 셀 문화 수집 렌즈 무료 비디오 현미경으로 수행 되 고 다음은 데이터를 4 단계 절차에 따라 분석: 다중 파장 홀로그램 재건, 셀 추적 셀 세분화 및 세포 분열 검출 알고리즘입니다. 결과적으로, 우리는 수집 하는 데이터 집합을 갖춘 10000 세포 주기 트랙과 2 x 10 이상6 셀 형태학 측정 보다는 더 가능 하다는 것을 보여줍니다.

Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

렌즈 무료 비디오 현미경 셀 문화 인큐베이터 안에 직접 모니터링할 수 있습니다. 여기 우리가 획득 하 고 배양된 HeLa 세포의 2.7 하루 종일 수집 분석 하는 데 사용 하는 전체 프로토콜 설명, 개별 셀 형태학 및 10584 세포 주기6 측정 추적 2.2 x 10의 데이터 집합에 지도.

동적에 대 한 렌즈 무료 비디오 현미경 및 부착 세포 배양의 정량 분석

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

lens-free-video-microscopy-for-dynamic-quantitative-analysis-adherent

Nanyang Technological University

Research

JoVE Journal

Biology

9.4K 조회수.  Université Grenoble Alpes. 렌즈 무료 비디오 현미경 셀 문화 인큐베이터 안에 직접 모니터링할 수 있습니다. 여기 우리가 획득 하 고 배양된 HeLa 세포의 2.7 하루 종일 수집 분석 하는 데 사용 하는 전체 프로토콜 설명, 개별 셀 형태학 및 10584 세포 주기6 측정 추적 2.2 x 10의 데이터 집합에 지도.

비디오: Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

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

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic 분석 : 자기편 셀

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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. ...

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, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

주변 줄기 세포 이주의 분석을위한 마우스 Cremaster 근육에있는 미세 혈관의의 intravital 현미경

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP 현미경을 이용한 miRNA의 gap junction 의존적 전달 분석

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

총 내부 반사 형광 현미경 검사법에 의해 살아있는 세포에 있는 세포 표면 수용체의 올리고머화 역학 은 수 및 밝기 분석과 결합됩니다

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...