이 작품은 NIH R01 GM47607, CA163296 및 CET에 NSF 1,334,493에 의해 지원됩니다. 우리는 원고의 중요한 읽기 터너 실험실의 구성원을 감사드립니다. 이 보고서에 표시된 모든 데이터는 Wormer 등. 2014 (11)의 허가에 의해 재현되었다.

Durotaxis 챔버 1. 준비 <ol><li> 하나의 6- 웰 조직 배양 플레이트를 준비하기 위해, 50ml의 원뿔형 튜브 용기를 설정. 50ml의 튜브에서 PDMS 염기 용액의 약 10g을 칭량 (솔루션은 매우 점성). </li><li> 90 : 1 기판 (1 ~ kPa로의 적합성), 필요에 가교 결합제 용액의 정확한 양을 결정 (90)에 의해 튜브에 PDMS 염기 용액의 중량을 측정 나눈다. 같은 튜브에 PDMS의 가교제 용액의 계산 된 금액을 추가합니다. 예 : 10g / 90 = 0.11 G. PDMS 염기 용액에 PDMS 가교제 용액 0.11 g을 추가한다. 참고 : 기본 및 가교제 솔루션은 모두 PDMS 키트에 제공됩니다. </li><li> 격렬 작은 주걱을 사용하여 실온에서 5 분 동안 PDMS 기재 / 가교 결합제의 혼합물을 혼합한다. 이 단계에서 혼합물을 공기 방울 다수를 포함 할 것이다. </li><li> BUB을 제거하기 위해 실온에서 50 XG에 5 분 동안 벤치 탑 원심 분리기에서 PDMS 기판을 원심 분리기bles. 이 경우 여전히 다시 5 분, 원심 분리 후 거품. </li><li> 90 피펫 1ml의 : 조직 배양의 각 웰에 1 PDMS 기판은 6 웰 플레이트를 처리 하였다. PDMS에 존재하는 임의의 잔류 기포는 21 G 바늘을 사용하여 팝핑함으로써,이 단계에서 제거 될 수있다. PDMS가 아니라 30 분 동안 전파하도록 허용합니다. </li><li> 종기 12mm 유리 # 5 분간 증류수 1 커버 슬립. 두 번 반복하고 증류수 된 커버를 저장합니다. </li><li> 부드럽게 한 후 PDMS에 커버 슬립을 드롭 PDMS 솔루션에 coverslip에의 한 쪽을 터치하여 조직 배양 플레이트의 각 웰에 하나, 건조 coverslip에 배치합니다. 커버 슬립이 침전으로, PDMS는 커버 슬립의 가장자리를 통해 잠식하기 시작하지만, 완전히 포함되지 않습니다. 이것은 (, B를 그림 1A 참조) 경화 후 PDMS와 유리 사이의 인터페이스를 생성합니다. </li><li> PDMS를 치료 (경화) 16 시간 동안 오븐에서 70 ° C에서 접시를 품어. 작은지면 배치세포 배양 후드에서 UV 및 E를 10 분 동안 살균. 참고 : 사용의 몇 일 이내에 판을 만드는 것이 가장 좋습니다. 그러나 판은 버퍼없이 파라 필름에 싸여과 품질에 띄는 감소없이 최대 2 주 동안 4 ℃에서 저장 될 수있다. </li></ol> 2. 셀 도금 주 : siRNA를 매개 녹다운의 효과를 연구하는 경우, 제조자의 지시 나 선택의 세포 유형에 대한 최적의 프로토콜을 이용하여 최저을 수행한다. <ol><li> 각 코트는 37 ℃에서 1 시간 동안 칼슘 및 마그네슘이없는 / ml의 PBS에 피브로넥틴을 10 μg의 1 ml의 챔버 durotaxis. 대안 적으로, 피브로넥틴은 4 ℃에서 16 시간 동안 / ml의 PBS에 피브로넥틴을 10 μg의 PDMS와 인큐베이션하여 분석 전에 일을 적용 할 수있다. 전체 표면을 PBS 용액에 피브로넥틴에 빠져들되어 있는지 확인합니다. </li><li> 열 변성 1 % BSA를 준비합니다. 0.5 g의 무게 BSA 및 PBS 50 ㎖에 용해.필터는 12 분 동안 80 ℃에서 0.22 ㎛의 필터와 열교환을 통해 살균 용액. 참고 :이 솔루션 4 ℃에서 도금 및 저장 셀하기 전에 하루를 준비합니다. </li><li> 피브로넥틴 솔루션을 기음과 PBS로 3 회 반복한다. 각 웰에 PBS에 열 변성 BSA 1 ㎖를 추가하고 실온에서 30 분 동안 품어. </li><li> durotaxis 챔버가 열 변성 BSA로 차단하는 동안를 Trypsinize 및 선택의 세포를 계산합니다. </li><li> 플레이트 원하는 특정 세포 유형에 필요한 매체를 사용 durotaxis 챔버의 각 웰에 2 ㎖를 1 × 105 세포. 세포 부착 및 5 % CO 2, 37 ℃에서 가습 된 배양기에서 4 시간 동안 기판에 도포 할 수있다. 주 : U2OS 세포는 일상적 2 mM L- 글루타민, 1 mM 피루브산 나트륨, 10 IU / ml의 페니실린, 10 μg의 / ml의 스트렙토 마이신이 보충 된 10 % FBS DMEM으로 유지된다. 주 :이 예에서 사용되는 세포 수에 최적화U2OS 세포. 다른 유형의 세포에 대한 최적화가 요구 될 수있다. 이 밀도는 세포를 다른 세포와 중요한 상호 작용없이 마이그레이션 할 수있는 충분한 공간을 제공합니다. </li></ol> 3. 라이브 셀 이미징 <ol><li> 10X 목표와 위상 콘트라스트를 사용하여, 거꾸로 현미경에 라이브 세포 이미징을 수행합니다. 현미경은 장기 촬상 동안 37 ℃ 온도의 제어 및 5 % CO 2 있도록 밀폐 환경, 습한 챔버가 장착되어야한다. </li><li> 세포를 약 3.5 시간 동안 분산 한 후, 현미경 실 플레이트를 조립한다. 샘플 (S)이 30 분 동안 챔버에서 평형화 허용한다. </li><li> 가능한 경우 현미경에 방문 자동화, 멀티 포인트를 설정합니다. (90) 사이의 인터페이스에 초점 : 1 PDMS와 유리 coverslip에 모든 durotaxis 챔버 당 40 점의 평균과의 인터페이스 주위에 이미지에 포인트를 선택합니다. 이미지 셀 최대 16 시간 동안 매 10 분. 참고 : 재관심의 기온은 커버 슬립의 가장자리와 PDMS와 커버 슬립 사이의 실제 인터페이스에 대응하는 내부 라인에 해당하는 외부 줄, 두 줄로 표시됩니다. 그림 1B를 참조하십시오. </li></ol> 4. 데이터 분석 <ol><li> 표 1과 같은 스프레드 시트를 생성한다. </li><li> 생성 된 각각의 동영상에서 유리 표면과 그 반대로 PDMS에서 교차 이벤트의 수를 카운트. 엑셀 스프레드 시트의 해당 열에 교차 이벤트의 수를 기록한다. 주 : 교차 이벤트가 어느 한 방향으로 PDMS와 유리 사이의 경계 위에 전달 세포핵으로 정의된다. </li><li> 다중 교차를 정량화하기 위해, 셀은 인터페이스 교차 회수를 카운트. 그 숫자는 셀이 영화의 단부에 위치 된 기판 상에 대응하는 엑셀 열에 기록되어야한다. t의 인터페이스를 가로 지르는 모든 셀에 대한 분석을 반복그는 영화. 촬영하는 동안 시야에서 마이그레이션 세포를 제외합니다. 주 : 세포가 피브로넥틴 - 코팅 된 PDMS와 유리 커버 슬립의 표면에 균일하게 부착 할 수있는 것을, 실험의 초기에 셀 카운팅함으로써 확인하는 것이 중요하다. </li><li> 유리 표면 (즉, durotaxis을 시행)에 PDMS에서 마이그레이션 세포의 비율을 계산합니다. 소프트에서 하드 및 교차 이벤트의 총 수에 의해 하드 및 분할에 끝났 여러 교차 이벤트에 이벤트를 건너의 수를 추가합니다. </li><li> 교차점의 총 수에 의해 다중 교차의 수를 분할하여 복수의 횡단의 비율을 계산한다. </li></ol>

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The authors have no conflicts to disclose.

여기에서 우리는 세포를 마이그레이션하는 durotaxis을 연구하는 간단한 분석을 설명합니다. 이 분석의 주요 장점은 PDMS를 사용 durotaxis 챔버 제조의 용이성이다. 기판의 강성을 용이하게 분석에서 다양한 강성의 연구를 허용하도록하기 위해 가교제 PDMS 염기 용액의 비율을 변경함으로써 조작 될 수있다. 그러나, 시스템의 하나의 잠재적 인 한계는 더 정교한 시스템 (4)에 의해 제공되는 강도...

세포 외 기질 (ECM)의 콜라겐, 피브로넥틴 및 라미닌을 포함하고, 가교 구조 단백질의 복잡한 배열로 구성된다. 이 아니라 ECM 셀룰러 조직 중요한 구조적지지를 제공한다는 확립되어 있지만, 세포가 활발히 세포 생존, 분화 및 세포 이동을 포함한 다양한 세포 과정을 조절하는 그들의 ECM 환경에서 물리적 인 변화에 대응 것을 나타내는 증거가 증가하고있다. 딱딱한 (~ 25 kPa로) 기판 조골 세포 분화를 촉진 한 예를 들어, ECM의 강성 차이는 연질 기판 (~ 1 kPa의) 신경성 계통 촉진과 함께, 서로 다른 계통으로 중간 엽 줄기 세포를 구동 할 수있다. 마찬가지로 기질 매트릭스 강성 증가는 주변 조직에 2,3- 유방 상피 세포 및 종양 침윤을 촉진하는 것으로 나타났다. 이 mechanosign의 특히 흥미로운 양태세포가 더 견고한 기판 4,5- 향해 우선적으로 마이그레이션하는 durotaxis로 알려진 프로세스에서 aling 활동 결과. 세포는 지속적 ECM 결합 인테그린 수용체를 통해 세포 외 환경의 물리적 특성을 감지. 이것은, 차례로, 국소 유착 또는 초점 콘택트 -6,7-라고도 접착제 구조의 형성을 구동 할 그들의 세포질 도메인 수많은 구조 및 시그널링 단백질의 축적을 촉진한다. 인테그린이 더 고유의 효소 활성이 없기 때문에, 신호는 변화하는 환경에 8 셀의 응답을 조정하기 위해 이러한 액세서리 단백질을 통해 ECM에서 릴레이됩니다. 따라서, mechanosignaling durotaxis 및 조절에 관여하는 주요 단백질의 동정 및 특성 규명은 수사의 중요한 영역이다. 다양한 모델 시스템 durotaxis 관내 연구 개발되어 왔지만, 대부분은 할이용 콜라겐 코팅 된 폴리 아크릴 아미드 기판 (4). 그러나, 폴리 아크릴 아미드의 기판의 제조는 기술적으로 어려울 수 있으며, 상기 분석 방법에 사용되는 콜라겐은 기판 (9)와 화학적으로 가교되어야한다. 폴리 디메틸 실록산 (PDMS) 기판은 폴리 아크릴 기판 (10)에 필적하는 기계적 성질을 나타내는 것으로 밝혀졌다. 그러나, PDMS 기판을 간단히 가교제 염기의 혼합 비율에 의해 제조되며, 이러한 기판은 이에 PDMS 세포 행동에 강성의 영향을 연구하기 쉬운 도구를 만드는 화학적 가교의 필요성없이 ECM 단백질로 코팅 될 수있다. 여기서, 우리는 부드러운 PDMS 기판이 경질 유리 커버 슬립과 통합되는 간단한 durotaxis 실을 준비하는 방법에 대해 설명합니다. 분석은, 아래에 설명 된대로, durotaxis을 연구하는 빠르고 간단한 방법을 제공합니다. 이 연구를 위해 우리는 siRNA를 매개로 결합 된 인간의 U2OS의 골육종 세포를 사용cdGAP의 최저은 durotaxis (11)이 초점 접착 단백질의 역할을 연구합니다. 중요한 것은,이 프로토콜은 개별적인 요구에 쉽게 적응 될 수있다. 다른 유형의 세포는 U2OS 세포를 대체 할 수 있고, 어떤 단백질은 뜨거나 durotaxis 동안 셀의 동작에 미치는 영향을 결정하기 과발현 될 수있다. 더욱이,이 프로토콜은 역학 거동 사용 FRAP 또는 접근법 FRET을 분석 형광 태그로 단백질을 포함하도록 구성 될 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

세포 외 매트릭스의 조성과 기계적 성질이 조직 유형 사이에서 매우 가변적이다. 이 크게 결합 조직 기질 다이버 영향 셀 동작은 세포 증식, 분화, 접착 시그널링 및 이동 방향을 포함하여 정상 및 병리학 적 과정을 조절한다. 이와 관련하여, 특정 세포 유형의 타고난 능력 durotaxis라고도 단단, 이하 호환 매트릭스 기판을 향해 이동한다. 이 현상은 배아 발생, 상처 복구 및 암세포 침윤 동안 중요한 역할을한다. 여기, 우리는 폴리 디메틸 실록산 (PDMS) 기판을 사용하여 시험 관내에서, durotaxis을 연구하는 간단한 분석을 설명합니다. 챔버 durotaxis 설명의 준비는 상대적으로 부드러운 PDMS 젤 단단한 유리 커버 슬립 사이에 강성 인터페이스를 만듭니다. 제공된 예에서, 우리는 cdc42의 역할을 입증하는 이들 durotaxis 챔버를 사용하고 / RAC1는 GTPase 활성 보호 자전거cdGAP에서, mechanosensing 인간 U2OS의 골육종 세포에서 durotaxis 규제. 이 분석은 mechanosignaling과 durotaxis에서 각각의 역할을 탐구하는 다른 세포 유형 및 / 또는 관심의 다른 단백질의 최저에 쉽게 적응할 수있다.

durotaxis 챔버의 개략도는도 1a에 도시되어있다. 소프트 PDMS 기판 (90 : 솔루션 가교제 PDMS 기재 1 혼합물)을 6 웰 접시에서 확산되고 유리 커버 슬립시켜 인터페이스를 생성하고 부분적으로 커버 슬립의 상부 표면을 덮는 PDMS, 위에 배치되는 다른 준수의 두 기판 사이. 소프트 PDMS 기판의 강성은 유리의 강도가 약 1 GPa의 1-2 반면, 뇌 조직의 일반적인 적합성에 필적 ~ 1 kPa로한다. PDMS...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

평가 셀 Mechanosensing 및 Durotaxis에 대한 단순화 된 시스템<em&gt; 체외</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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Universidad de Cartagena UDEC

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JoVE Journal

Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.1K 조회수.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

비디오: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

향상제 시퀀스를 평가하기위한 모자이크 Zebrafish의 Transgenesis

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

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Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

체외 세포 이동 및 내습 분석 실험에서

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

식물의 셀없는 시스템에서 프로 테오 저하 시금

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

1 채널의 세포 부착 된 패치 클램프 녹음

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

내피 세포 접착 정량<em&gt; 체외</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

평가 셀 Mechanosensing 및 Durotaxis에 대한 단순화 된 시스템

요약

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이 비디오의 챕터

향상제 시퀀스를 평가하기위한 모자이크 Zebrafish의 Transgenesis

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Ribosome 바운드 초기 Polypeptides의 분리

체외 세포 이동 및 내습 분석 실험에서

식물의 셀없는 시스템에서 프로 테오 저하 시금

1 채널의 세포 부착 된 패치 클램프 녹음

내피 세포 접착 정량

실험실 주택의 청록색 송사리 (Nothobranchius furzeri)에 대 한 프로토콜