Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

이산화 티탄 1. 준비 유리 커버 슬립을 피복 된 <ol><li> 번호 다이아몬드 스크 라이버를 이용하여 커버 슬립. 이것은 각 커버 슬립 추적 할뿐만 아니라, 시료의 정확한면이 위를 향하게되도록뿐만 돕는다. 제 피라니아 용액에 침지하여이를 다음, DDH 2 O 흐르는, 커버 슬립을 청소 (H 2 SO 4 : H 2 O 2 = 4 : 1). 10 분 후, 2 DDH O.에서 철저 8 번 커버 슬립 린스 N이 흐름에 따라 커버 슬립을 건조. </li><li> 무선 주파수 (RF) 스퍼터링 시스템에 이산화 티탄 목표를 설정합니다. 폴리이 미드 테이프를 사용하는 스퍼터링 장치의 샘플 홀더에 부착 된 커버를. 스퍼터링 챔버에 샘플 홀더를 놓습니다. 압력이 2.0 × 10에 도달 할 때까지 실을 대피 -4 아빠를. </li><li> 챔버 내에 Ar 가스를 도입하고 4.0 mTorr로에 증착 압력을 설정합니다. 셔터 폐쇄 유지하면서 서서히 증가(70) W.에 RF 전력 </li><li> 셔터를 열고 120-150 나노 미터 (도 1 : 단계 A)의 두께의 필름을 얻기 위해 15 분 동안 스퍼터. 피막의 성장 속도는 각각의 시스템에 대해 유도 될 필요가있다. </li></ol> 세포 발수 필름 2. 표면 코팅 <ol><li> 플라즈마 반응기의 제조에 의해 제공되는 지시에 따라, O 2 플라즈마에 의해 시료를 처리하여 이산화 티탄 표면을 친수성으로. 우리는 100 SCCM의 O 2 흐름 (W) (200)에서 5 분의 샘플을 취급합니다. DDH 2 O에서 샘플을 담그고 표면이 초 친수성인지 확인합니다. 철저 N이 흐름에 따라 표면을 건조시킵니다. </li><li> 100 ml의 톨루엔 39.6 μL OTS를 추가하여 1 MM의 옥타 데실 트리클로로 실란 (OTS) 솔루션을 준비합니다. RT에서 1 시간 동안 용액의 시료를 담그. N 2 - 채워진 장갑 가방 (: 단계 B 그림 1) 내부에이 단계를 실시한다. </li><li> physisorbed 분자를 제거하려면, sonicat전자 톨루엔, 아세톤, 에탄올 샘플, 그 순서대로 5 분 각각에 대한 DDH 2 O. 신선한 DDH 2 O에서 샘플 네 번 씻어 N 2의 흐름에 따라 표면을 건조. 표면 100-110 °의 접촉각과 소수성이어야한다. </li></ol> 3. 현지 외 표면 패터닝 <ol><li> 층류 후드에서 작업, 표면에 다이아몬드 스크 라이버 여러 긁힌 자국을 그립니다. 마크 처리 된 영역을 추적하고 또한 초점 현미경을 가져에 도움이됩니다. 5 분 동안 70 % 에탄올에 침지시켜 샘플을 OTS 코팅 이산화 티탄 살균. 그런 다음 멸균 DDH 2 O 두 번 샘플을 씻어 </li><li> 35 mm의 접시에 샘플을 놓고 PC12 성장 매체 2 ㎖를 추가 (단계 4.2 참조). CO 2 배양기에서 3 시간 이상 (37 ° C)에 대해 품어. 이 절차는 혈청 알부민은 표면 상에 흡수하도록하기위한 것이다. 흡착 된 알부민은 다른 보호 자전거의 후속 흡착을 억제기능과 세포 (그림 1 : 단계 C). </li><li> 기다리는 동안, 반전 형광 현미경을 설정합니다. <ol><li> 아크 램프를 켜고 UV 필터 큐브를 삽입하고 20 배에 대물 렌즈를 설정합니다. </li><li> 광 강도 I (W cm -2)을 사용하여 UV 강도 측정기를 측정하고 (J의 형상 -2)에서 D의 투여 량 (초) 조사 시간 t를 계산한다 : t = D / I. 예를 들어, 200 J의 cm의 도즈 량 조사하는 -2 Mw가 600 cm -2의 광원을 사용하여 333 초간 조사가 필요하다. </li><li> 스테이지 마이크로 미터를 사용하여 영역의 크기가 예를 들면 200 μm의, 조사되도록 설정하는 필드 조리개를 닫는다. </li></ol></li><li> (; 최종 농도 300 μg의 ml의 골 -1-IV)에서 3 시간 배양 후, 3.0 mL의 밀리그램 -1-IV 형 콜라겐 200 μL와 매체를 보충. </li><li> 현미경 단계로 35 밀리미터 접시를 전송합니다. 스크래치 M 찾기아크는, 샘플 표면에 초점 현미경, 200 J의 cm -2 (도 1 스텝 D)의 용량으로 UV 광을 조사한다. UV 조사의 영역은 필드 조리개의 개구를 조정함으로써, 또는 중재 형상의 메탈 마스크 필드 조리개를 대체함으로써 변경하거나 할 수있다. </li><li> (골-IV)없이 새로운 성장 매체와 매체를 교체하고 다시 인큐베이터에서 샘플을 놓습니다. </li></ol> 4. 세포 배양 <ol><li> PC12 세포의 루틴 배양 콜라겐 코팅 된 플라스틱 접시에 수행된다. <ol><li> 코트 골-IV와 60 mm의 조직 배양 접시에 먼저 DDH 2 O로 표면을 적시고 모든 DDH 2 O를 대기음 </li><li> 300 μg의 용액을 준비 -1 골-IV를 DDH 2 O (3 mg의 용액 1) 10 배 원액을 희석하여 </li><li> 300 μg의 ml의 -1 60 밀리미터 접시 당 골-IV의 200 μl를 추가합니다. 표면을 통해 확산 솔루션을 보자. </li><li> 드라이 T그는 약 1 시간 동안 층류 후드에서 요리. </li><li> 둘 베코 인산염 완충 식염수 (D-PBS) 4 ㎖로 두 번 표면을 씻어. 바로 코팅 된 접시를 사용하지 않을 경우, 2 DDH O. 4 ㎖로 한번 씻어 접시 DDH 2 O 흡입 한 후, 인큐베이터에서 접시를 저장합니다. 이 2 주 이상 보관 유지하지보십시오. </li></ol></li><li> PC12 세포로 구성 성장 배지에서 성장시킨다 : 둘 베코 변형 이글 배지 (저 글루코즈) + 10 % 소 태아 혈청 + 5 % 말 혈청 + 1 % 페니실린 - 스트렙토 마이신 용액. CO 2 배양기에서 세포를 품어 (37 ℃, 5 % CO 2), 그리고 매일 매체의 절반을 교체합니다. 통로 세포는 그들이 합류에 도달하기 전에. <ol><li> 흡 인물은 성장 배지 및 추가 2ml의 PBS + (D-PBS + 10 mg을 -1 ml의 소 혈청 알부민 (BSA) + 10 mM의 EDTA)을 37 ℃로 데워진. 37 ℃에서 5 분 동안 배양한다. 부드럽게 CE를 분리 접시를 누릅니다LLS. </li><li> 15 ML 원뿔 튜브에 세포를 수집합니다. 37 ℃로 데워진 신선한 D-PBS의 3 ㎖와 접시를 씻어. </li><li> 4 분 동안 150 × g에서 원심 분리 튜브. </li><li> 상등액을 흡인하고, 성장 배지 1 ㎖를 추가한다. </li><li> 1~3 : 루틴 배양, 세포 1 분할 5. </li></ol></li><li> , UV-수정 OTS / 이산화 티탄 샘플 세포를 플레이트 세포 밀도를 계산하고 35 밀리미터 접시 (그림 1 단계 : E) 3.0 × 10 5 세포를 추가합니다. 1-2일위한 가습 인큐베이터 접시 (37 ° C를, 5 % CO 2) 배양한다. 나이브 모두 신경 성장 인자 (NGF) -differentiated PC12 세포 패터닝 실험에 사용될 수있다. NGF 차별화, 7S-NGF NG 100 ml의 시료 -1에 도금 전에 세포 성장 배지 며칠에 첨가된다. NGF 분화는 PC12 세포의 점착력을 증가시키는 것, 특히 시츄 패터닝 E에서 용이하게 취급xperiments. </li></ol> 5. 원위치 표면 패터닝 <ol><li> 전 현장 표면 개질에 문화 1 ~ 2 일 후, 세포 부착 및 자외선 조사 지역에서만 성장하고 있는지 확인합니다. 단계 3.4에 설명 된대로, 현미경을 설정합니다. </li><li> 성장 배지를 함유하는 새로운 35-mm 조직 배양 접시에 시료를 이동시켜, (NGF-분화 된 세포를 사용하는 경우) 100 NG ml의 -1 NGF, 및 100 μg의 용액 -1 골-IV. </li><li> 현미경의 무대에서 접시를 놓습니다. 적절한 위치를 찾아 200 J 센티미터 -2 (그림 1 단계 : F, G)의 용량으로 자외선을 조사. 새로 생성 허용 영역은 그림 1에서 별표로 표시됩니다 : 단계 G. </li><li> 30 분 내에 단일 샘플의 처리를 완료하려고합니다. 조사 후, 골-IV없이 다시 매체에 샘플을 전송합니다. </li><li> CU와 함께 요리를 운반 할 때는 특히 조심ltured 세포 접시에서 샘플을 전송은 패턴 화 된 세포의 분리 방지하기 위해 접시. </li></ol><img alt="그림 1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> 전체 프로세스의 도식 그림을 그림. 자세한 내용은 텍스트를 참조하십시오. <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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Nanotech. 13, 213-218 (2015).</li><li>Arima, Y., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Surface+Functional+Groups+on+Protein+Adsorption+and+Subsequent+Cell+Adhesion+using+Self-Assembled+Monolayers.">Effects of Surface Functional Groups on Protein Adsorption and Subsequent Cell Adhesion using Self-Assembled Monolayers.</a> J. Mater. Chem. 17, 4079-4087 (2007).</li><li>Fujishima, A., Zhang, X., Tryk, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TiO2+Photocatalysis+and+Related+Surface+Phenomena.">TiO2 Photocatalysis and Related Surface Phenomena.</a> Surf. Sci. Rep. 63, 515-582 (2008).</li><li>Sigal, G. B., Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Surface+Wettability+on+the+Adsorption+of+Proteins+and+Detergents.">Effect of Surface Wettability on the Adsorption of Proteins and Detergents.</a> J. Am. Chem. Soc. 120, 3464-3473 (1998).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+and+Photo-Patternable+Superhydrophobic+Film.">A Transparent and Photo-Patternable Superhydrophobic Film.</a> Chem. Commun. 2007, 4949-4951 (1039).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Hippocampal+Neurons.">Culturing Hippocampal Neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li></ol>

The authors have nothing to disclose.

현재의 프로토콜에서, 이산화 티탄 막을 RF 마그네트론 스퍼터링에 의해 형성 하였다. 그것은 우리가 재현 서브 나노 거칠기 광촉매 이산화 티탄 필름을 제조 할 수 있기 때문에 우리는 증착이 방법을 선호. 스퍼터 증착 공정은 재료 과학자 및 전자 엔지니어 잘 알고 있지만, 생물 학자에 매우 액세스 할 수 없습니다. 이 경우에, 스핀 코팅 이산화 티탄 필름 (23)의 ?...

반도체 리소그래피 공정 및 그 유도체 - 포토 리소그래피 1,2, 전자빔 리소그래피 3-6 및 7-10 인쇄 마이크로 콘택트로서 - 이제 정의 된 위치 및 형상에 살아있는 세포가 성장하는 세포 생물학에서 확립 도구가되었다. 패터닝 방법은, 비 허용 세포에서 배경 허용 코팅 마이크로 섬 이루어진 미세 가공 된 기판의 사용에 의존한다. 이러한 기판 패턴 세포에 템플릿 역할을합니다. 이러한 기술은 우리에게 세포의 고유 특성을 추출하고, 세포 기반 약물 검사 (11)의 처리량을 증가시키기 위해, 단일 및 다중 세포 수준에서 세포와 그 기능을 설계 할 수있는 새로운 방법을 제공하고 있습니다. 템플릿 패턴 형상이 즉, 현장에서 변경 될 수 있다면 세포의 배양 동안 자유도 셀 패턴에서 크게 증가 할 것urface. 그들은 분위기 또는 진공에서 샘플을 처리 이후 패턴 형성 방법은 종래의 직접 여기에 적용 할 수 없다. 따라서 여러 새로운 표면 개질 기술들은 단지 몇 가지 이름을, 광 반응성 화합물 (12, 13) 또는 레이저 어블 5,14에, 예를 들면, 기준이되는, 제안되어있다. 제안 된 방법은 잘 최 등. (16)와 나카니시 (17)에 의해 최근에. Robertus 등의 알 15을 검토하고있다. 다음은이 문서에서, 우리는 이산화 티타늄에 유기 분자의 광촉매 분해의 활용에 현장 표면 개질 (이산화 티탄) 표면 (18, 19)의 새로운 프로토콜을 설명합니다. 이 방법에서, 이산화 티탄 막은 유리 기판과 세포 인터페이스 유기 막 사이에 삽입되고, 상기 유기 막 국소 자외선 (UV)을 조사하여 그 자리에서 분해되어관심 영역 (λ &lt;388 ㎚)에 빛. 우리는 새로운 프로토콜은 세포 외 기질 단백질과 살아있는 세포 모두 외부 계와 시츄 미세 패턴을 생성하는데 사용될 수 있음을 보여준다. 이산화 티탄은 세포 배양 실험에서 소개가 친절하게 기능이있는, 생체 적합성 화학적으로 안정하고, 광학적으로 투명하다. 이 프로토콜은 세포 배양 환경에서 세포 배양 지지체를 수정하기위한 재료 과학 기반의 대안을 제공합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass coverslip</td>
			<td>Warner Instruments</td>
			<td>CS-15R15</td>
			<td>15 mm, #1.5 thickness</td>
		</tr>
		<tr>
			<td>Diamond scriber</td>
			<td>Ogura Jewel Industry</td>
			<td>D-Point Pen</td>
			<td></td>
		</tr>
		<tr>
			<td>RF sputtering system</td>
			<td>ANELVA</td>
			<td>SPC350</td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 sputtering target</td>
			<td>Kojundo Chemical Lab</td>
			<td>Titanium (IV) oxide, target</td>
			<td>Purity, 99.9%</td>
		</tr>
		<tr>
			<td>Plasma reactor</td>
			<td>Yamato</td>
			<td>PR301</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octadecyltrichlorosilane 
			(OTS)</td>
			<td>Aldrich</td>
			<td>104817</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Wako</td>
			<td>204-01866</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (35 mm)</td>
			<td>Greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (60 mm)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-IV collagen</td>
			<td>Nitta Gelatin</td>
			<td>Cellmatrix Type IV</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td>Heat-inactivate and pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td>Pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100x)</td>
			<td>Nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>7S nerve growth factor (NGF)</td>
			<td>Alomone Labs</td>
			<td>N-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo</td>
			<td>N001</td>
			<td>Stock solution in 0.5 M</td>
		</tr>
		<tr>
			<td>TiO2 nanoparticle</td>
			<td>Tayca</td>
			<td>TKD-701</td>
			<td></td>
		</tr>
	</tbody>
</table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

이산화 티타늄의 표면 (이산화 티탄)에 흡착 된 유기 오염 물질은 자외선 (UV) 광 하에서 광촉매 작용에 의해 분해 될 수있다. 여기에서 우리는 로컬 기판 표면의 세포 친 화성을 ​​변경할 수있는 이산화 티탄 광촉매를 사용하는 새로운 프로토콜을 설명합니다. 이 실험을 위해, 얇은 이산화 티탄 막은 스퍼터 - 코팅 유리 커버 슬립이며, 이산화 티탄 표면은 후속 적으로 세포 부착을 억제 옥타 데실 트리클로로 실란 (OTS)로부터 유도 된 단일 층 오가 노 실란으로 개질 하였다. 샘플은 세포 배양 배지에 침지하고, UV 광이 각형 영역에 집중 조사 하였다. 신경 세포주 PC12 세포 샘플에서 도금 될 때, 셀은 UV 조사 된 영역에 부착. 우리는 상기 셀이 상기 기판 상에 성장하는 경우에도,이 표면 개질도 즉, 인 시츄로 수행 될 수 있음을 보여준다. 표면의 적절한 수정은 세포 외 기질 P 필요콜라겐 rotein하면 UV 조사시의 중간에 존재한다. 여기에 제시된 기술은 잠재적 하에서 배양 세포를 공 배양 시스템을 구축하거나 임의로 조작 패터닝 여러 세포 유형에 이용 될 수있다.

도 2a는 스퍼터 - 증착 된 이산화 티탄 필름의 단면 주사 전자 현미경 (SEM) 이미지를 나타낸다. 관찰로부터, 필름의 두께는 약 150 nm 인 것으로 추정되었다. 여기에 띄는 증착 이산화 티탄 막의 평탄성이다. 원자력 현미경 (AFM)에 의한 추가 분석은면의 제곱 평균 제곱근 (RMS) 조도는 0.2nm의 (도 2B)이한다고 밝혀졌다. 이산화 티탄?...

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

산화 티타늄을 사용하여 수성 환경에서 단백질과 세포를 포토 패터닝<sub&gt; 2</sub&gt; 광촉매

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

Research

JoVE Journal

Bioengineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

7.8K 조회수.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

비디오: Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

핵산, 단백질과 작은 분자의 Reagentless 감지를위한 전기 -의 DNA Biosensors의 제작

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

연구

생체공학

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

부정적 얼룩 EM에 의해 단백질과 Macromolecular의 단지를 떠올리 : 그리드 준비에서 이미지 수집

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

3 차원 매트릭스에서 찬란 태그가 추가된 단백질을 표현 마이 그 레이션 전지의 라이브 세포 이미징

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

수성 환경에서 탄화수소 전환을 사용하는 툴킷

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

RootChip를 사용하여 루트 환경의 신속한 조작과 Arabidopsis 루트 성장의 시간 저속 형광 이미징

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

수성 2 단계 시스템을 사용하여 셀 공동 문화 패턴

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

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 ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

효소 Microreactors 및 그들의 전자기 작동기로 사용 하는 수성 방울

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

패터닝 단백질과 세포의 다목적 방법

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...