이러한 연구 노력은 화학, 생물, 환경, 보너스 번호 CBET-0853780 및 CBET-1159344 및 건강의 국립 연구소, 환경 건강 과학 국립 연구소 (NIEHS)에 따라 전송 (CBET) 시스템의 국립 과학 재단 (National Science Foundation) 부문에 의해 지원되었다 보너스 번호 1R43ES022567-01, 그리고 국립 암 연구소, 보너스 번호 CA127745-01에 따라 암 이미징 프로그램에 따라. 이 연구에서 사용 된 IVIS 루미나 계기는 미국 육군 국방 대학 연​​구 계측 프로그램에서 얻은 것입니다.

1. 세포의 준비 <ol><li> 액체 질소 냉동 재고 발광 HEK293 세포의 병을 복구하고 Dulbecco의 수정 이글의 10 % 태아 소 혈청과 보충 배지 (DMEM), 0.01 mM의 불필요한 아미노산, 1X 항생제 항진균 및 T 0.01 밀리미터 나트륨 피루브산에서 그들을 성장 37 75 조직 배양 플라스크 ° C와 5 % CO 2. 참고 : 미디어 및 보조 구성 요소는 세포 라인에 따라 달라질 수 있으므로 그에 따라 선택해야합니다. </li><li> 새로 고침 중간마다 도달 할 때까지 2~3일 ~ 80 % 합류. 참고 : 필요한 세포의 양이 실험 설계를 기반으로합니다. 필요한 경우, 더 많은 세포를 계대 배양하여 얻을 수 있습니다. </li><li> 테스트를 위해 수확 세포. <ol><li> 매체를 제거하고 인산염 세포를 씻어 부드럽게 플라스크를 소용돌이 후 폐기 PBS를 소비하여 식염수 (PBS)를 버퍼. </li><li> 2 분 동안 37 ° C에서 트립신과 부화를 추가또는 셀 때까지 플라스크에서 분리했다. </li><li> PBS에서 분리 된 세포를 수집하고 깨끗한 원심 분리기 튜브로 전송할 수 있습니다. 참고 : nonadherent 세포 라인을 원심 분리하여 별도의 셀을 사용하고 PBS로 한번 세척하는 경우. </li></ol></li><li> hemacytometer 또는 다른 세포 계수 시스템을 사용하여 전체 세포 수를 결정합니다. </li><li> 5 분 300 XG에 세포 현탁액을 원심 분리기. 뜨는을 취소하고 신선한 prewarmed 매체에 세포를 resuspend을. </li><li> 불투명 멀티 웰 플레이트의 각 우물에 세포의 씨앗 같은 수. 이 예에서는, 플레이트 5 × 10 블랙 24 - 웰 플레이트의 각 웰에 1 ML 볼륨에서 5 세포. 판 세중 우물에서 매체의 동일한 볼륨은 대조군 역할을합니다. 참고 : 각 우물에서 도금 세포의 수는 유연하고 개별 실험 최적화 할 수 있습니다. 각 발광 세포 라인에 대한 실험적 세포 수와 biolumi 사이의 관계를 결정nescent 출력뿐만 아니라, 최소 검출 셀은 이전의 모든 독성 시험을 계산합니다. 표준 이미징 조건에서 인구 크기의 넓은 범위 (예를 들어, 1 개의 10월 3일부터 1일까지 X 10 6 세포)에 걸쳐이, 측정 생물 발광을 할 수 있습니다. </li><li> 아래에 설명 된대로 시험 화합물 (들)과 세포를 치료. </li></ol> 2. 화학 준비 <ol><li> 농축 원액으로 테스트되고있는 화학 물질을 준비합니다. 이 예에서는 100 ㎎ / 블레오 마이신 계열의 항생제의 ML 주식 솔루션을 사용합니다. 참고 : 유기 용매가 화학 물질 추가하기위한 수단으로 사용하는 경우 특히, 잠재적 인 차량 기반의 독성을 최소화하기 위해, 그것은 주식 농도가 적어도 1,000 배에서 원하는 포스트 응용 프로그램의 농도 것이 좋습니다. </li><li> 직접 세포에 준비된 화학 주식을 추가합니다. 이 예에서는, 복용량 최종 100 농도, 200, 300, 그리고 트라이에서 400 ㎍ / ㎖에서 관심 항생제우물 plicate. 컨트롤로 치료 세포의 세 개의 우물을 남겨주세요. 참고 : 대상 화학 물질의 최종 농도는 특정 실험 목표에 따라 선택해야합니다. 농도의 넓은 범위는 일반적으로 독성 효과의 전체 스펙트럼을 얻기 위하여 시험된다. </li></ol> 3. 이미징 및 데이터 분석 <ol><li> 즉시 화학 물질뿐만 아니라 다음, 이미지 수집 및 생물 발광 측정에 적합한 기기의 이미징 챔버에 접시를 놓습니다. </li><li> 각 읽기 10 분 통합 시간을 사용하여 24 시간 동안 측정 생물 발광은 매 15 분. 참고 :이 예제에 사용되는 적분 시간 당 플레이트 기준이며, 선택의 다른 luminometers를 사용하여 각도 단위로 측정 조정할 수 있습니다. 통합 시간은 선택한 발광 세포 라인에 따라도 조정할 수 있습니다. 생물 발광은 세포 파괴 나 외부 자극없이 자율적으로 제작되고 있기 때문에 읽기INGS는 특정 실험 목적을 달성하기 위해 원하는 시점에서 찍은 수 있습니다. </li><li> 호환 소프트웨어를 사용하여 관심 영역을 식별하여 각 우물에서 발광 강도를 정량화. 하나 광속 (광자 / 초) 또는 각 샘플의 평균 래디언스 (광자 / CM / 초 2 / steridian)와 같은 광 출력을 표시합니다. </li></ol>

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DM 닫기, SA RIPP 및 GS Sayler 490 생명 공학, 주식 회사의 설립자 및 소유자이다

이 방법은 살아있는 세포가 지속적으로 자신의 일생을 통해 모니터링 할 수 있습니다 체외 독성 스크리닝 검사에서와 같은 자율적으로 발광 포유 동물 세포를 사용하는 방법을 보여줍니다. 이 프로토콜은 유연하고 필요에 따라 특정 실험 조건에 맞게 수정할 수 있습니다. 예를 들어, 실험은 급성 독성을 추적에 적합 여기서 제시하지만, 반복적으로 증가 시간 간격 (예 : 매 24 ?...

그들은 식품 의약청 (Food and Drug Administration) 소비자의 사용이 승인되기 전에 미국에서, 인간의 소비를위한 의약품 및 기타 제품은 광범위한 평가를 필요로합니다. 이 테스트를 수행하기위한 재정 부담이 크게 소비자를위한 비용 증가로 새로운 화합물을 개발하기 때문에 번역의 비용을 증가 개발자 1에 배치됩니다. 이 검사 전통적으로 많은 인간의 호스트에 대한 프록시 역할을하는 동물의 과목을 활용했지만,이 (독극물 / ADME 흡착, 분배, 대사, 배설 및 독성에 연간 소요 추정 2,800,000,000달러로, 큰 재정 부담이 될 입증되었습니다 ) 단독 심사 2과 동물 모델을 안정적으로 인간 학적 반응에게 3 예측할 수없는 것을 제안 증거 확보. 따라서 체외 인간의 세포 배양 기반의 테스트 때문에 상대적으로 지난 20 년 동안 인기를 얻고있다비용, 높은 처리량, 그리고 인간의 생체 이용률 및 독성 4의 좋은 표현입니다. 현재 셀 문화 기반의 독성 스크리닝 방법은 세포 생존에게 5을 평가하기 위해, 세포막의 무결성을 프로빙, 또는 미토콘드리아 활동의 수준을 추적, 내생 가능한 세포질 효소의 활동을 심사, 이러한 ATP 수준의 측정 등 다양한 엔드 포인트를, 고용 6. 측정함으로써 단일 시점의 데이터를 생성, 촬영하기 전에 그러나,에 관계없이 선택된 엔드 포인트의이 방법은 모든 시료의 파괴​​를 필요로합니다. 결과적으로 많은 양의 샘플은 준비 및 기본 독성 동력학 연구를위한 병렬 처리, 다시 새로운 화합물 개발에 필요한 비용과 노동력에 추가해야합니다. 또한, 사용 분석은 Gaussia 루시 페라 제 7 Vargula 루시 페라 제 8 및 Metridia 루시 페라 제 9과 루시 페라 분비세포 용해에 대한 필요성을 제거하고 엔드 포인트 측정을위한 미디어의 일부만을 필요로하지만, 이들은 여전히​​ 일정 시간 지점에서 샘플링을 제한하고 또한 외생 빛을 활성화 기판의 추가를 필요로하는 개발되었습니다. 필요한 샘플 파괴의 손해뿐만 아니라 기판의 비용을 제거하기 위해를 방지하기 위해, 인간의 세포 라인은 비슷합니다 살아있는 세포를 지속적으로 모니터링 할 수 있도록 전체 세균의 생물 발광 (LUX) 유전자 카세트 (luxCDABEfrp) 표현하는 설계되었습니다 형광 염료 기반의 라이브 셀 이미징하지만, 추가 광자 활성화 및 현미경 조사 절차없이. 이 세포주 따라서 시료의 파괴​​를 방지, 구조적으로 외부 자극에 대한 필요없이 지속적 직접 검출을위한 광 신호를 생성 할 수 있습니다. 기작이 세포에서 생성되는 발광 신호가 발생의 luxAB 구성된 루시 페라 제 효소는 감소 리보플라빈 인산염의 존재 (FRP 유전자에 의해 FMN에서 재활용 FMNH 2의 긴 사슬 지방산 알데히드 (내생 기판을 사용하여 luxCDE 유전자 산물에 의해 합성 및 재생)의 산화를 촉진시 제품)와 산소 분자 10. 숙주 세포에있는 럭스 카세트의 발현 따라서 생산 세포의 파괴 또는 외인성 기판 추가하지 않고 감지 할 빛을 수 있습니다. 마찬가지로, 럭스 유전자와 내생 사용할 수 FMN, 및 O 2 cosubstrates 및 FMNH 2 FMN의 변환을 지원 할 수있는 환경의 정비에 대한 요구 사이의 상호 작용 결과 생물 발광 신호가 단지 생활에서 감지 할 수 있도록 , 대사 활성 셀. 이러한 요구 사항은 이전에 해당 럭스-BAS를 보여 악용되었습니다ED 발광 출력은 세포 인구 크기 11 그 독성 화합물에 노출 용량 - 반응 패션 12 autobioluminescent 생산을 저해와 강하게 연관시킵니다. 여기에서 우리는 독성 테스트를위한 autobioluminescent 포유 동물 세포의 응용 프로그램을 확인하는 대표적인 예로 알려진 DNA 손상 활동 블레오 마이신 계열의 항생제의 자동 독성 스크리닝을 보여 이전에 특징 autobioluminescent 인간의 배아 신장 (HEK293) 세포 라인 (11)을 사용합니다.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

포유류 세포 기반 생체 분석에 널리 독성 연구를위한 동물 실험의 대안으로 채택되었지만 인해 반딧불 루시 페라 기반 검사 방법의 파괴 특성에 필요하게되는 병렬 샘플 준비의 높은 금융 및 시간 비용으로 인해 제한되어있다 . 이 비디오는 그 화합물의 세포 독성 효과를 모니터링하기위한 저렴하고 손쉬운 방법으로, 루시페린 기판의 파괴적인 추가를 필요로하지 않는 자율적으로 발광 포유 동물 세포의 활용을 설명합니다. 안정적 전체 세균의 생물 발광 (luxCDABEfrp) 유전자 카세트를 표현 포유 동물 세포는 자율적으로 광 신호를 생성하는 외부 에너지 원, 또는 전통적 시료의 파괴에 의한 비용과 가능한 간섭 루시페린 기판, 여기의 첨가없이 490 nm에서 피크 광학 이미징 절차 동안 수행의. 외부 자극으로부터이 독립 결과 신호 만 활성화 신진 대사 감지 될 것을 의미, 전적으로 셀 발광 반응을 유지하기위한 부담을 배치합니다. 발광 생산의 변화가 세포의 성장과 신진 대사에 악영향을 나타내는 때문에이 특성은 럭스 발현 세포주 세포 독성 효과에 대한 biosentinel로 사용하기위한 최상의 후보합니다. 마찬가지로, 필요한 샘플 파괴의 자율적 인 성격과 부족은 독성 물질 노출의 기간 동안 실시간으로 동일한 샘플의 반복 이미징을 허용하고 자동화 된 방식으로 기존 영상 장비를 사용하여 여러 샘플을 통해 수행 할 수 있습니다.

본 연구에서는 autobioluminescent HEK293 세포의 역학은 항생제 노출 (그림 1)에 대한 응답으로 24 시간 동안 지속적으로 감시 하였다. 에 바인딩하고 DNA 13 쪼개서 살아있는 세포를 죽이는 것으로 알려져 블레오 마이신 가족의 구성원이 항생제의 독성 효과는 사색 이미지를 직접 시각화 할 수있는 치료 세포에 비해 발광 생산 감소를 통해 입증되었다 (그림 2). 다양한 처?...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

세균의 생물 발광 유전자 카세트를 표현하는 포유 동물 세포 (<em&gt; 룩스</em&gt;) 자율적으로 빛을 생산하고 있습니다. 화학 물질에의 노출에 따라 결과 발광 역학은이 세포에게 쉽게 높은 처리량 자동화에 적용 할 수있는 저렴하고, 연속, 실시간 독성 검사 도구를 만드는 세포의 성장과 신진 대사에 치료 효과를 반영하기 위해 입증되었습니다.

세포 독성의 지속적인 실시간 모니터링을위한 자율적으로 바이오 루미 네 센트 포유 동물 세포

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

autonomously-bioluminescent-mammalian-cells-for-continuous-real-time

Research

JoVE Journal

Biology

10.0K 조회수.  Oak Ridge National Laboratory. 세균의 생물 발광 유전자 카세트를 표현하는 포유 동물 세포 ( 룩스) 자율적으로 빛을 생산하고 있습니다. 화학 물질에의 노출에 따라 결과 발광 역학은이 세포에게 쉽게 높은 처리량 자동화에 적용 할 수있는 저렴하고, 연속, 실시간 독성 검사 도구를 만드는 세포의 성장과 신진 대사에 치료 효과를 반영하기 위해 입증되었습니다.

비디오: Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

포유 동물 세포를 Trypsinizing 및 Subculturing

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

연구

생물학

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

정량 실시간 PCR (qPCR) 배열을 사용하여 사전 마이크로 RNAS과 microRNAs의 프로 파일링

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

형광 공명 에너지 전송과 리간드 - 수용체 상호 작용의 실시간 모니터링

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

Unimolecular 바이오 센서를 사용하여 라이브 세포에서 이벤트를 신호의 실시간 모니터링을위한 현미경을 안달

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

형광 현미경으로 여러 세대 이상 라이브 포유류의 세포에 플라스미드 복제 모니터링

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

포유 동물 세포에서 헴 합성 레벨 측정

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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

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

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

프로브 기반의 실시간 PCR은 마이크로 RNA의 정량 측정을위한 접근 방법

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

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

박테리아, 효모 및 인간 세포에서 일시적으로 형성된 분자 샤페론 어셈블리의 시투 모니터링

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...