이 작품은 (DEC에) R35GM119617을 부여 토마스 F.와 케이트 밀러 제프리스 Memoria (DEC에) 신뢰와 NIH에 의해 지원되었다. 공 초점 현미경 영상은 VCU 나노 물질 특성 코어 (NCC) 시설에서 수행되었다.

1. Nesprin-2G 센서 DNA 및 다른 플라스미드 DNA를 구합니다 <ol><li> 상업적인 소스로부터 Nesprin 2G-TS (인장 센서) Nesprin HL-2G (헤드리스) 제어 mTFP1 금성 및 TSmod를 구합니다. 모든 DNA 플라스미드를 전파 이전 12,13 바와 같이, 이러한 DH5-α 표준 E. 콜라이 균주를 사용하여 정제. </li></ol> Nesprin-2G 및 기타 플라스미드 DNA 2. 형질 세포 <ol><li> 온도 (37 ° C) 및 CO 2 5 %로 규제 표준 세포 배양 인큐베이터에서 6- 웰 세포 배양 접시에 70-90 %의 합류점에 NIH 3T3 섬유 아세포의 세포 성장. 세포 성장 매체의 경우, 10 % 소 태아 혈청 둘 베코의 변형 이글 중간 (DMEM)을 사용합니다. </li><li> 세포 배양 후드에서, 배지를 제거하고 감소 된 혈청 배지 세포의 약 1 mL로 각 웰을 씻어. 각 웰에 감소 혈청 세포 매체의 800 μL를 추가상기 인큐베이터에서 6- 웰 챔버를 배치했다. </li><li> 지질 담체 용액 35 μL와 함께 1.5 ㎖의 튜브에 감소 된 혈청 세포 배지 700 μL 피펫은 &quot;lipomix&quot;를 형성한다. 피펫으로 섞는다. 에 &quot;L.&quot;로 튜브 라벨 </li><li> 여섯 개 1.5 ㎖의 튜브를 수집하고, 각 관으로 감소 된 혈청 세포 배지 6 μL 피펫 (100)을 통해 1 라벨. <ol><li> mVenus 항 피펫 1 μg의 튜브에 튜브 (3) 및 제 피펫 1 mTFP의 μg의로 튜브 1 및 2 피펫 2 Nesprin-HL의 μg의 한 단계 1 피펫 2 Nesprin 2G-TS의 μg의으로부터 플라스미드 DNA의 농도를 사용하여 튜브 (6)에 다시 사용하지 마십시오 피펫 팁을 DNA의 다른 유형을 피펫 팅 할 때. </li></ol></li><li> 각 분류 관 (1-6)에 &quot;L&quot;튜브로부터 lipomix 100 μL 피펫 및 피펫 팅을 반복하여 혼합한다. 각 튜브에 대한 깨끗한 피펫을 사용합니다. 10 ~ 20 분 동안 품어. </li><li> 70-90 % 합류로 6 웰 챔버의 웰에 각각 분류 관에서 200 μL를 추가세포. 해당 DNA와 각 웰의 상부에 라벨했다. 4-6 시간 동안 인큐베이터에서 6- 웰 챔버를 놓는다. </li><li> 배지를 흡인하고 린스 감소 된 혈청 배지 셀 1-2 mL를 첨가. 상기 감소 된 - 혈청 배지를 세포 기음 각 웰에 트립신 2 mL를 첨가하고, 인큐베이터 (5-15 분)로 6 웰 접시를 배치했다. </li><li> 세포 배양기에서 분리되지만, 피막 (6) 유리 바닥은 인산 완충 용액 (PBS)에 용해 된 20 ㎍ / ml의 농도에서 피브로넥틴의 층으로 요리를보고. 코팅하는 요리를 세포 배양 후드 (약 20 분)의 표면을 허용한다. </li><li> 세포가 분리되면 DMEM 2 용액을 첨가함으로써 트립신을 중성화. </li><li> 또한 표지 된 15 mL의 원심 분리 튜브에 각각의 콘텐츠를 전송하고 스윙 로터 원심 분리기에서 5 분 동안 90 XG에서 스핀 다운. 상등액을 흡인 피펫 혼합하여 DMEM 1000 μL의 각 세포 펠렛을 다시 일시. </li><li> 로부터 피브로넥틴 혼합물 기음유리 접시 피펫 유리 접시 상에 각 세포 현탁액을 1000 μL. </li><li> 세포가 유리 접시의 바닥 (~ 15 분)에 정착 한 후, 세포 배양기에서 잘 장소 각각 다른 DMEM 1 ㎖의 + 10 % FBS + 1 % 스트렙토 펜을 추가한다. 세포가 적어도 18 ~ 24 시간 동안 센서를 부착하고 표현 할 수 있습니다. 참고 : 세포 상업 양이온 성 지질 형질 전환 시약 (재료의 표 참조)을 사용하여 형질 전환된다. 대안 적으로, 안정한 세포주는 독소에 대한 내성을 부여하는 유전자를 가진 플라스미드를 사용하여 선택 될 수있다 (Nesprin-TS와 -hl 대한 pcDNA 벡터 플라스미드 네티 신 내성을 발현하는 세포 (물질의 표를 참조)를 제공하는 DNA 저장소 사이트에서 사용할 ). 또한 바이러스 감염 방법 (렌티 바이러스, 레트로 바이러스 또는 아데노 바이러스)를 형질 어렵다 셀 센서를 표현하는데 이용 될 수있다. </li><li> Nesprin-TS 외에도 Nesprin HL 제로위한 부가적인 세포를 형질CE 제어 2.1-2.12 단계에서 설명한 바와 같이, TSmod 또한 제로 - 힘 제어로 사용될 수 Nesprin HL-유사한 FRET을 발휘한다. </li><li> mTFP1 금성로 형질 세포 스펙트럼 지문 (단계 4 참조)를 생성한다. 참고 mTFP1 금성은 일반적 Nesprin-TS보다 높은 수준의 발현과 같은 DNA의 하부 양 유사한 발현 수준을 달성하기 위해 형질 전환 될 필요가있다. </li></ol> 3. 형질 전환 효율을 확인 <ol><li> 대략 18-24 시간 형질을 마친 후,도 셀 (통상 5-30 %)의 총 수에 형광 세포의 수를 비교하여 형질 전환의 효율을 조사하기 반전 형광 현미경을 사용한다. <ol><li> 462 나노 미터 (mTFP1) 또는 525 나노 미터 (금성) 및 492 nm의 (mTFP1) 또는 525 나노 미터 (금성) 근처에 중심 발광 필터 근처의 여기 주파수가 장착 된 형광 현미경을 사용한다. 참고 : 또는 GFP 필터 세트 mTFP1의 조합을 캡처했습니다 것NUS 배출량은 형질 전환 효율의 확인을 허용합니다. </li></ol></li><li> 형질 전환 후 48 시간 내에 이미지 살아있는 세포. <ol><li> 대안 적으로, 48 시간 후 8 PBS에서 5 분간, 상점, 볼 (칼슘 및 마그네슘과 PBS)에서 4 % 파라 포름 알데히드로 세포를 고정한다. 참고 : 48 시간을 넘어, 신호 품질 및 강도는 붕괴. 세포를 고정하기 (8)로 표현되는 FRET 포함한 상태를 유지; 매체를 장착하여 FRET (14)에 영향을 미칠 수있다 그러나, 셀은, PBS에 이미징되어야한다. 표정의 변화가있을 수에 따라 고정 셀은 다른 셀에 고정 비교 될 수있다. 주의 : 파라 포름 알데히드는 독성이있다. 적절한 개인 보호 장비 (PPE)를 착용 할 것. </li></ol></li></ol> 스펙트럼 Unmixing에 대한 mTFP1과 금성 형광 발색단 4. 캡처 스펙트럼 지문 <ol><li> 세포 배양 후드에서 촬상 mediu와 세포 배지를 교체10 % 소 태아 혈청 m (HEPES 완충). </li><li> 온도 제어 (37 ° C) 공 초점 레이저 주사 현미경 단계에서 보는 요리를 놓는다. </li><li> 1.4 개구 수 60X 배율 오일 대물 위에 mTFP1 형질 세포 유리 뷰잉 접시 놓는다. 주 : 오일 밀접 유리 기판의 굴절률이 유사 할 60X 대물 렌즈에 레이저 스캐닝 현미경에 사용된다. 오일은 대물 렌즈의 상단에 배치하고 유리 커버 슬립과 직접 접촉한다. </li><li> mTFP1는 458 nm의 여기 원 및 500 nm의 발광 중심 대역 통과 필터로 발현 세포를 찾아. </li><li> 시야에서 형광 셀 (여기에서 사용되는 소프트웨어 &quot;람다 모드&quot;) 스펙트럼 검출 모드를 선택하고, 분광 화상 (도 3-1)을 캡처; 10 개 나노 미터 단위 (도 3-3)을 이용하여 458 nm의 이후의 모든 주파수를 포함한다. 밝은 fluoresc를 선택셀 (20 화소 반경)에 ENT 영역 (ROI). 참고 : mTFP1 발현 투자 수익 (ROI)의 스펙트럼 형태는 셀에 걸쳐 상대적으로 일정하게 유지해야한다. 형상이 현저하게 변화되면, 레이저를 재조정하고, 신호 대 잡음비를 개선하기 위해 설정을 얻는다. </li><li> 클릭하여 스펙트럼 데이터베이스에, 형광 ROI 의미 정규화 강도를 추가 &quot;데이터베이스에 스펙트럼을 저장합니다.&quot; </li><li> 레이저 파워를 최적화하고 양호한 신호 대 잡음비가 성취되도록 얻는다. 설정은 다른 장비에 따라 다를 것입니다. 배경 기준으로 비 - 형광, 형질 감염되지 않은 세포를 사용하여, 그러나, 검출기 (포화)의 동적 범위를 넘어, 세포가 밝은 때까지 상기 이득과 전력을 증가시킨다. 배경 세포는 평균 후 감지 형광 없어야합니다. </li><li> 다음을 제외 금성 - 형질 전환 된 세포의 과정을 반복합니다 : <ol><li> 여기 주파수가 515 nm에서인지 확인하고, 밴드 패스 필터는 CE라고금성 발현 세포를 찾을 때 530 나노 미터 근처 ntered. </li><li> 의 &quot;스펙트럼 모드&quot;내지 515 대신 458 nm 인 여기 주파수를 사용한다. </li></ol></li></ol> 5. 캡처 혼합하지 않은 이미지 <ol><li> 에 캡처 모드 전환 &quot;스펙트럼 Unmixing을.&quot; </li><li> unmixing 채널로 (도 3, 화살표 2) 금성 mTFP1의 스펙트럼 지문을 추가한다. </li><li> 다시 458 nm의 아르곤 소스 여진 레이저를 설정한다. </li><li> 60X 오일 대물 위 Nesprin-TS 볼 접시 놓는다. </li><li> 충분히 밝은 식 형광 세포에 집중하면, 신호 대 잡음비를 최적화하는 게인 및 레이저 파워를 조정한다. 참고 Nesprin-TS의 FRET 효율은 20 % 가까이 있기 때문에, 혼합되지 않은 금성 화상이 섞이지 mTFP1 이미지보다 실질적으로 더 어둡게 될 것이다. 허용 전력 및 이득 설정이 반복적으로 결정되면, 이들 매개 변수는 모두 이미지 대위에 대한 일정하게 유지해야ured. </li><li> (모든 포화 화소 화상 처리 동안 제거되는) 비교적 유사한 밝기 Nesprin-TS 셀 15-20 이미지 캡처 최소 화소 과도한 포화를 방지. </li><li> 동일한 촬상 파라미터를 사용하여 HL-Nesprin 세포 촬상 처리를 반복한다. </li></ol> 6. 이미지 프로세싱 및 비율 이미지 분석 <ol><li> 사용하여 오픈 소스 ImageJ에 (피지) 소프트웨어 (http://fiji.sc/), BioFormats Reader를 사용하여 열려있는 기본 형식 이미지. </li><li> 전 과정을 이미지와는 이전에 설정된 프로토콜 15을 사용하는 비율 이미지를 계산한다. </li></ol>

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Biochem. 21 (5-6), 489-498 (2008).</li><li>Kardash, E., Bandemer, J., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+activity+in+live+embryos+using+fluorescence+resonance+energy+transfer+biosensors.">Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors.</a> Nat Protoc. 6 (12), 1835-1846 (2011).</li><li>Vaziri, A., Mofrad, M. R. 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U.S.A. 98 (14), 7765-7770 (2001).</li><li>Nagayama, K., Yahiro, Y., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+stabilize+the+position+of+intranuclear+DNA+through+mechanical+connection+with+the+nucleus+in+vascular+smooth+muscle+cells.">Stress fibers stabilize the position of intranuclear DNA through mechanical connection with the nucleus in vascular smooth muscle cells.</a> FEBS letters. 585 (24), 3992-3997 (2011).</li><li>Luxton, G. W. G., Gomes, E. R., Folker, E. S., Vintinner, E., Gundersen, G. 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A., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>LaCroix, A. S., Rothenberg, K. E., Berginski, M. E., Urs, A. N., Hoffman, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction,+imaging,+and+analysis+of+FRET-based+tension+sensors+in+living+cells.">Construction, imaging, and analysis of FRET-based tension sensors in living cells.</a> Methods Cell Biol. , (2015).</li></ol>

The authors have nothing to disclose.

방법 및 Nesprin-2G 핵 LINC 복잡 단백질 걸쳐 기계적 장력 생균 촬상 시연은 위에서 언급되었다. 이전이 연구에, 이러한 마이크로 피펫 흡인 세포 계측법, 자성 비드, 현미경 레이저 절제와 같은 다양한 기술들은, 세포핵에 변형을 적용하고, 벌크 물성 16, 17, 18을 측정하기 위해 사용되었다. 그러나, 최근의 작품까지, 어떤...

포스 성 유전자 부호화 FRET 센서 최근 생균 인장 계의 힘을 측정하는 단백질 1, 2, 3, 4에인가되는 방법을 기계적 힘에 대한 통찰력을 제공하기위한 중요한 도구로 떠오르고있다. 이러한 도구를 사용하여, 연구자들은 기존의 형광 현미경을 이용하여 살아있는 세포에서 비 침습적 영상 세포 내 힘 수 있습니다. 이러한 센서는 탄성 펩티드 (3)에 의해 분리 된 FRET 쌍 (도너와 억 셉터 형광 단백질, 자주 청색 공여체 및 수용체 노란색)로 구성된다. C- 또는 N- 말단 태그 대조적으로,이 센서는 분자 스트레인 게이지로서 행동하고,이 단백질에 걸쳐 전송되는 기계적인 힘을 측정 할 수있는 단백질의 내부 부위에 삽입된다. 프렛-P 사이의 증가 된 거리에서 센서 결과 걸쳐 기계적 장력을 증대공기 감소 FRET 3의 결과. 그 결과, FRET 반비례 인장력에 관한 것이다. 이러한 형광 기반 센서는 초점 접착 단백질 (vinculin 3 talin 4), 세포 골격 단백질 (α-티닌 5), 및 세포 - 세포 접합 단백질을 위해 개발 된 (E-cadherin의 6, 7, VE-cadherin의 8 및 PECAM 8). 이러한 바이오 센서에서 가장 자주 사용되는 잘 특징으로하는 탄성 링커 TSmod라고도하고 거미줄 단백질 flagelliform로부터 유도 된 40 개 아미노산 (GPGGA) (8)의 반복되는 시퀀스로 구성된다. TSmod은 인장력 3/5 PN 1 FRET에 응답하여 선형 탄성 나노 스프링처럼 행동하는 것으로 나타났다. flagelliform의 다른 길이는 동적 R을 변경하는 데 사용할 수 있습니다TSmod FRET 힘 감도 (9)의 플랜지. flagelliform 외에도 스펙 트린은 (HP35라고도 함) (5)와 villin 투구 펩티드 4는 유사한 포스 바이오 센서 (4) 사이의 FRET 쌍 탄성 펩티드로서 사용되어왔다를 반복한다. 마지막으로, 최근의 보고서는 TSmod는 압축력 (10)를 감지하는 데 사용할 수 있다는 것을 보여 주었다. 최근 TSmod 내생 비슷하게 동작 미니 Nesprin2G (도 2C)로 알려진 이전에 개발 된 절두 Nesprin2G 단백질 삽입 이용하여 nucleo - 골격 (LINC) 복합 단백질 Nesprin2G의 링커 힘 센서를 개발 Nesprin-2G (11). LINC 복합체는 핵에 얇은 골격을 연결하는 세포질, 핵의 외부에서 내부로 이끌어 다중 단백질을 포함한다. Nesprin-2G는 구조적 단백질에 결합되는 두세포질과 핵 주변 공간에 태양 단백질 액틴 세포 골격. 우리의 바이오 센서를 사용하여, 우리는 Nesprin-2G는 NIH3T3 섬유 아세포 2 액토 마이 오신에 의존 긴장 될 수 있음을 보여줄 수 있었다. 이것은 그 힘이 바로 핵 LINC 단지에 단백질을 통해 측정 한 처음, 그리고 제어 공학의 핵에 힘의 역할을 이해하는 중요한 도구가 될 가능성이 높습니다. 프로토콜은 아래 Nesprin-을 발현하는 세포의 FRET 화상의 포유 동물 세포에서 Nesprin 장력 센서 (Nesprin-TS)의 발현뿐만 아니라, 수집 및 분석을 포함하여 Nesprin-2G 힘 센서를 사용하는 방법의 상세한 방법을 제공한다 TS. 분광 검출기를 구비 한 반전 된 공 초점 레이저 주사 현미경을 사용하여, 발광 증감을 측정하는 방법을 설명 unmixing 스펙트럼을 이용하여 비율 적 FRET FRET 및 촬상이 제공된다. 출력 이미지가 상대적 비율 적 ...로서 확인하는데 사용될 수있다ntitative 힘 비교. 이 프로토콜은 섬유 아세포 Nesprin-TS의 발현에 집중되는 반면, 세포주 및 일차 전지 모두 포함한 다른 포유 동물 세포에 쉽게 적응할 수있다. 또한, 이미지 수집 및 FRET 분석에 관련된 프로토콜이 용이 다른 단백질에 대해 개발 된 다른 FRET 기반 포스 바이오 센서에 적용될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1183">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-TS DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68127</td>
			<td style="width:475px;">Retrieve from https://www.addgene.org/68127/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-HL DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68128</td>
			<td>Retrieve from https://www.addgene.org/68128/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mTFP1 DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">54613</td>
			<td>Retrieve from https://www.addgene.org/54613/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mVenus DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">27793</td>
			<td>Retrieve from https://www.addgene.org/27793/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">TSmod DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">26021</td>
			<td>Retrieve from https://www.addgene.org/26021/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Competent Cells</td>
			<td style="width:108px;">Bioline</td>
			<td style="width:119px;">BIO-85026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Liquid LB Media</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10855001</td>
			<td>https://www.thermofisher.com/order/catalog/product/10855001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Solid LB Bacterial Culture Plates</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Ampicillin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Spectrophotometer</td>
			<td style="width:108px;">Biorad</td>
			<td style="width:119px;">273 BR 07335</td>
			<td>SmartSpec Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">quartz cuvette</td>
			<td style="width:108px;">Biorad</td>
			<td align="right" style="width:119px;">1702504</td>
			<td>Cuvette for SmartSpec Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">&#160;DNA isolation kit</td>
			<td style="width:108px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740412.5</td>
			<td>NucleoBond Xtra Midi Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">6-well cell culture dish</td>
			<td style="width:108px;">Falcon-Corning</td>
			<td align="right" style="width:119px;">353046</td>
			<td>Multiwell 6-well Polystrene Culture Dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Dulbecco&#39;s Modified Eagle Medium, (DMEM) cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td>DMEM(1x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16170-078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">reduced serum cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>Reduced Serum Medium, &#34;optimem&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Lipid Carrier Solution</td>
			<td style="width:108px;">invitrogen</td>
			<td style="width:119px;">11668-019</td>
			<td>Lipid Reagent, &#34;Lipofectamine 2000&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">1.5mL sterile plastic tube</td>
			<td style="width:108px;">Denville</td>
			<td style="width:119px;">c2170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Trypsin</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>0.25% Trypsin-EDTA (1x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">glass-bottom microscope viewing dish</td>
			<td style="width:108px;">In Vitro Scientific</td>
			<td style="width:119px;">D35-20-1.5-N</td>
			<td>35mm Dish with 20mm Bottom Well #1.5 glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Fibronectin</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">33016015</td>
			<td>fibronectin human protein,plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">14190-144</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">15mL sterile centrifuge tube</td>
			<td style="width:108px;">Greiner bio-one</td>
			<td align="right" style="width:119px;">188261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">swinging rotor centrifuge&#160;</td>
			<td style="width:108px;">Thermo electron</td>
			<td style="width:119px;">Centra CL2</td>
			<td>Swinging rotor thermo electron 236</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">cell culture biosafety hood</td>
			<td style="width:108px;">Forma Scientific</td>
			<td align="right" style="width:119px;">1284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;climate controlled cell culture incubator</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">3596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">inverted LED widefield fluorescent microscope</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">EVOS FL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Clear HEPES buffered imaging media</td>
			<td style="width:108px;">Molecular Probes</td>
			<td style="width:119px;">A14291DJ</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Fetal bovine Serum</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:483px;">Temperature Controlled-Inverted confocal w/458 and 515nm laser sources&#160;</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:119px;">&#160;LSM 710-w/spectral META detector</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Outgrowth Media</td>
			<td style="width:108px;">Newengland Biolabs</td>
			<td style="width:119px;">B9020s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">NIH 3T3 Fibroblasts</td>
			<td style="width:108px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

전술 한 프로토콜에 따라, 플라스미드 DNA는 DNA 저장소로부터 취득하고, 대장균에 형질 전환. 센서 DNA를 발현하는 대장균을 LB / 암피실린 플레이트로부터 선택 LB 액체 배지에서 증폭 하였다. 벡터의 증폭에 이어, 플라스미드 DNA는 표준 시판 DNA 분리 키트를 이용 TRIS-EDTA 버퍼로 정제 하였다. 분광 광도계를 사용하여, 정제 된 DNA는 ㎍ / ㎖ (도 1a, 1-2 ...

Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

포스터 공명 에너지 전달 (FRET) -force의 바이오 센서를 사용하는 프로토콜은 핵 LINC 단지에서 기계의 힘을 측정하는

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

a-protocol-for-using-frster-resonance-energy-transfer-fret-force

Research

JoVE Journal

Bioengineering

10.5K 조회수.  Virginia Commonwealth University. A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex. 

비디오: A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

연구

생체공학

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

솔루션의 생물 고분자의 작은 및 와이드 앵글 X-레이 산란 연구

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

정량은 SENP1 단백 분해 효소 반응 속도론 결정에 대해 (포스터 공명 에너지 전송) 분석 안달

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

조직 공학을위한 이축 기계 로딩 생물 반응기의 설계

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

원자 힘 현미경을 사용하여 살아있는 세포의 기계적 특성을 측정

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

생활 효모 세포에서 미토콘드리아 산화 환원 상태와 ATP를 측정 비례 바이오 센서

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.

The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.

복셀에서 지식 : 복잡한 전자 현미경 3D-데이터의 분할에 대한 실용 가이드

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

마이크로 기둥 미세 유체 장치에서 바이오 필름 보리 형성을위한 프로토콜

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

로봇 미세 접촉 인쇄 (R-μCP)를 사용하여 복합 문화 기판 및 순차 친 핵성 치환 제조

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...