우리는 최적화 및 프로토콜을 검증하기 위해 기여 우리의 실험실에있는 모든 사람들에게 감사합니다. 이 작품은 INSERM과 국가 연구 기관 (ANR-09-JCJC-0116 및 ANR-11 BSV1-033-02)에 의해 지원되었다. ET는 고등 교육과 연구의 프랑스 내각에서 연구 교제 그녀의 박사 과정에 지원되었다.

솔루션 1. 준비 <ol><li> PBS 14.5 ml의 37 % 포름 알데히드 5.5 mL로 희석하여 PBS (저염)에서 10 %의 포름 알데히드를 준비한다. PBS 50 ㎖ 중의 글리신 3.8 g을 용해시켜 1 M 글리신, pH가 8.0을 준비한다; 1X PBS 100 mM의 글리신을 얻기 위해 상기 용액을 희석. </li><li> 1X PBS에서 0.1 % 트리톤 - X100을 준비합니다. 3.0 M 염화 나트륨, 25 ℃에서의 pH 7.0 탈 이온수 버퍼 제조 0.30 M 시트르산 삼 나트륨을 이용하여 20 배 염 구연산 나트륨 (SSC)를 준비한다. 1x에서이 버퍼를 희석 탈 이온수를 사용하여 0.01x. </li></ol> 세포의 2. 고정 참고 : 본 연구에서 HuH7 간암 세포주를 사용하지만,이 방법은 다른 접착 세포 배양에 적용 가능하다. <ol><li> 코팅 35 mm의 유리 바닥 접시 당 150,000 세포에서 (10 % 소 태아 혈청 0.01 % 페니실린 - 스트렙토 마이신 재고 보충 DMEM 1g / L의 포도당에서 배양) 플레이트 HuH7 세포. 일차 전지에 작업 할 때문화, 콜라겐 코팅 요리를 사용합니다. </li><li> 다음 날, 배지를 제거합니다. PBS와 대기음 1 ml의 세포를 씻으십시오. 포름 알데하이드 10 % 1 ㎖를 첨가하여 세포를 고정하고, 교반하에 실온 (RT)에서 10 분 동안 배양한다. </li><li> 1 M 글리신 1 ㎖로 반응을 중지하고 회전에 의해 섞는다. 반응 정지 액을 제거하고 1X PBS 1 ㎖를 첨가하여 세포를 세척; 회전 및 흡인에 의해 선동. 세포에 100 mM의 글리신의 1 ML을 추가하고 15 교반하면서 실온에서 분 후 흡인 그들을 품어. 주 : 여기에서의 프로토콜은 중지 될 수 있고, 다음 단계는 또 다른 일 연기 될 수있다. 이 경우, 100 mM의 글리신 1 ㎖를 추가하고, 필요한 경우, 4 ℃에서 보관. </li></ol> 세포의 3 Permeabilization <ol><li> 15 교반하면서 실온에서 분 후 흡인 부화, 1X PBS에서 0.1 % 트리톤 - X100의 1 ML을 추가합니다. 일차 전지의 cultur 작업시 20 분 -이 배양 시간은 15까지 증가 될 수있다ES (예를 들어, 기본 쥐 간세포). 1X PBS 1 ㎖를 첨가하여 세포를 씻어; 대기음. </li></ol> 4. 차단 <ol><li> (키트에서 제공) 각 샘플에 차단 솔루션의 40 μl를 추가; 이 부피는 샘플을 커버하기 위해 증가 될 수있다. 습도 챔버에서 37 ° C에서 30 분 동안 요리를 부화. </li><li> 요리의 전원 차단 솔루션을 누릅니다. 이 재현성에 영향을 미칠 것 같이, 각 슬라이드에 대해 동일한 잔류 볼륨을 얻기 위해 시도합니다. 샘플이 건조하게하지 마십시오! </li></ol> 5. 차 항체 <ol><li> 1X PBS의 기본 항체를 희석과 요리 (VDAC1 마우스 항체 : 1/100, IP3R1 토끼 항체 : 1/500)의 솔루션을 추가 할 수 있습니다. 또한, GRP75 또는 CypD 항체 (1/500에서 사용 모두 마우스 항체) 대신 VDAC1 사용할 수 있습니다. </li><li> 4 ℃에서 습도 챔버에서 밤새 품어. 0.01 % 트윈 (TBS-T)와 슬라이드를 트리스 완충 식염수를 사용하여 두 번 씻으십시오. </li></ol><p클래스 = &quot;jove_title&quot;&gt; 6. 근접 내고 분석 프로브 <ol><li> 근접 결찰 검정 프로브는 키트가 제공된다. 일차 항체의 종류에 따라 프로브를 선택합니다. </li><li> 항체 희석액에 5 : 두 개의 근접 내고 분석 프로브 1을 준비합니다. 혼합물을 실온에서 20 분 동안 앉아 할 수 있습니다. 희석 근접 내고 분석 프로브 솔루션을 추가합니다. 37 ° C에서 1 시간 동안 예열 된 습도 챔버 내에서 요리를 부화. TBS-T로 요리 두 번 씻으십시오. </li></ol> 7. 내고 <ol><li> 현장 검출 시약 텍사스 빨간색 키트에 사용합니다. </li><li> 고순도의 물에 5 잘 섞는다 : (키트에서 제공)을 5 배 결찰 1 개를 희석. 1시 40분 및 와류 (키트로 제공)를 1X 라이 게이션 용액으로 희석 리가. 즉시 샘플뿐만 아니라 이전까지 리가 아제를 추가 할 때까지 기다립니다. </li><li> 미리 가열 습도 참에 슬라이드를 각각의 샘플이 용액 (35 mm 유리 바닥 요리 40 μl를) 추가 및 배양BER 37 ℃에서 30 분. TBS-T로 슬라이드 두 번 씻으십시오. </li></ol> 8. 증폭 참고 :주의, 빛에 민감한 시약한다. <ol><li> 고순도의 물에 5 : (키트에서 제공)을 5 배 증폭 1 개를 희석. 냉동 블록을 사용하여 냉동실에서 중합 효소를 분리합니다 (-20 ° C). (키트에서 제공) 중합 효소 1 배 증폭 솔루션과 소용돌이에서 1:80 희석. 혼합물을 사용하기 전에 즉시 중합 효소를 추가합니다. </li><li> 각각의 샘플이 용액 (35 mm 유리 바닥 요리 40 μL)를 추가합니다. 37 ℃에서 100 분 동안 예열 된 습도 챔버에 슬라이드를 부화. 슬라이드의 오프 증폭 - 중합 효소 솔루션을 누릅니다. </li></ol> 9. 최종 세척 <ol><li> 2 분 동안 1 배 SSC 세척 버퍼에 슬라이드를 씻으십시오. 2 분 동안 0.01x SSC 세척 버퍼에 슬라이드를 씻으십시오. 요리는 어둠 속에서 실온에서 건조 시키십시오. </li></ol> 이미징 10. 준비 <ol&gt; <li> 공기 방울이 커버 슬립 아래에서 발견되지 얻을 보장 (수성 및 응고되지 않음) DAPI를 포함하는 설치 매체의 최소한의 볼륨을 사용하여 슬라이드를 탑재합니다. 매니큐어 가장자리를 밀봉하기 위해 사용될 수있다. (: 594 nm의, 방출 : 624 nm의 배율 : 63X 여기) 형광 또는 공 초점 현미경을 사용하여 분석하기 전에 약 15 분 동안 기다립니다. </li><li> 촬상 후, 어둠 속에서 -20 ° C에서 슬라이드를 저장한다. </li></ol>

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The authors declare that they have no competing financial interests.

Collectively, our studies indicate that the in situ proximity ligation assay is truly a relevant strategy to follow and quantify endogenous ER-mitochondria interactions in fixed cells, without the need for using organelle-specific fluorophores or fluorescent proteins. The specific use of VDAC1/IP3R1 antibodies has been adapted to study ER-mitochondria interactions in HuH7 cells. However, alternative isoforms of VDAC and IP3R may be used, depending on the cell type. In this case, antibodies need to be validated b...

미토콘드리아 소포체 (ER)은 세포 소기관 독립적 아니지만 미토콘드리아 관련 소포체 막 (MAM)로 정의 접촉 부위에서 구조적 및 기능적으로 상호 작용한다. 사실, MAMS은 양쪽에서 단백질 간의 상호 작용을 가능하게하여 ER의 세포막과 미토콘드리아가 밀접하게 나란히 놓이는되는 지역에 해당합니다. 그럼에도 불구하고,이 세포 소기관의 멤브레인은이 지역 내에서 융합하지 않는, 그래서 그들은 자신의 별도의 기관을 유지한다. MAMS은 에너지 대사 및 세포 생존 1-3에 영향을 미치는, 미토콘드리아에 ER에서 칼슘의 중요한 역할 (칼슘)과 인지질 전송을한다. 응급실과 미토콘드리아 사이의 관계는 제 1 전자 현미경으로 1970 년대에 가시화되었다. 그 이후, 투과 전자 현미경 4,5-, 6,7- 전자 단층 또는 ER 및 미토콘드리아 특정 형광의 면역 지역화S / 형광 단백질 (8)는 고전 ER-미토콘드리아의 상호 작용을 연구하는 데 사용되었다. MAM의 분석을위한 다른 유용한 도구는 세포 이하 분획의 사용에 기초한다. 이것은 퍼콜 구배 (9)에 결합 된 차동 초 원심 MAM 분획을 분리 할 수있다. 그러나, 최종 제품은 풍부한 MAM 분획이 아닌 순수한 분획을 포함한다. 전부,이 전략은 특히 민감한 및 / 또는 정량적하지 않습니다, 그들은 큰 심사에 쉽게 의무가 없습니다. 다르게는, 약물 - 유도 성 형광 간 소기관 링커를 사용하여 유전 학적 접근이 등장 있지만 단백질 10의 내인성 발현 수준 소기관 상호 작용의 분석을 허용하지 않는다. MAM 11의 IP3R / GRP75 / VDAC 복잡 Szabadkai의 발견에 기초하여, 우리는 ER-미토콘드리아의 상호 작용을 분석하는 정량적 방법을 개발 하였다. 우리는 현장 근접 ligati에 사용분석에 감지하고 VDAC1 및 IP3R1 사이의 상호 작용을 정량화하고, 고정 셀 (12)의 MAM 인터페이스에서 칼슘 2 + -channeling 복잡한에 관여하는 두 소기관 표면 단백질. 간단히 말해서, 우리는 ER 막에서 외부 미토콘드리아 막 (마우스 항 - VDAC1 차 항체) 및 IP3R1 (토끼 항 IP3R1 차 항체) (그림 1, 패널)에서 VDAC1을 프로빙. 그리고, 상기 분석에있어서, 우리는 항 - 마우스와 상보적인 올리고 뉴클레오티드로 확장되어 접합 항 - 토끼 IgG를 (마우스 및 토끼 근접 결찰 분석법 프로브) 모두를 추가했다. 두 개의 표적 단백질이 40 내지 이하의 거리에있는 경우, 올리고 뉴클레오티드는 원형의 DNA를 주형 (도 1, 패널 B)의 형성을 허용하도록 연속적으로 첨가 커넥터 올리고와 혼성화 할 수있다. 이 원형 DNA 분자에 공유 변위 센서 중 하나에 장착 된 단일 가닥 DNA 생성물 생성 결찰 증폭된다 (도 1, 패널 c) </stro&gt; NG. MAM 계면에서 ER 및 미토콘드리아 간의 거리 내지 6 근접 결찰 증폭 인해 텍사스 레드 표지 된 올리고 뉴클레오티드 프로브 (도 1, 패널 (D)의 혼성화에이어서 검출 선도 할 수있는 (25)는 10 nm 내지 때문에 ). 각 형광 점 따라서 개별 셀의 현장에서 ER-미토콘드리아 상호 작용의 정량화를 허용 VDAC1 / IP3R1 사이의 상호 작용을 나타냅니다. <img alt="그림 1" src="/files/ftp_upload/54899/54899fig1.jpg" /> 그림 1 : 제자리 근접 내고 분석에 의해 소포체 - 미토콘드리아 상호 작용의 검출의 개략도. a) VDAC1 및 MAM의 계면 근방에서 그 에피토프에 결합 할 수 IP3R1 향한 토끼 차 항체에 대해 유도 마우스 일차 항체 b) 인접 결찰 프로브 쌍의 또마우스와 토끼 IgG에 대해 지시했다. 이 프로브는 커넥터 올리고의 결찰에 대한 템플릿을 형성 할 수있는 DNA 가닥을 첨부했습니다. c) 결찰 후 형성된 원형의 DNA 가닥이 텍사스 레드 표지 된 올리고 뉴클레오티드를 이용하여 형광 현미경으로 도트 시각화) D 증폭 될 수있다. <a href="http://ecsource.jove.com/files/ftp_upload/54899/54899fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 시츄 근접 결찰 분석 실험에서 유사한 항체 GRP75 / IP3R1 쌍뿐만 아니라 시클로 필린의 D (CypD) 수행 될 수있다 / IP3R1 항체 CypD는 MAM 계면에서 IP3R / GRP75 / VDAC 복합체와 상호 작용하는 것으로 나타났다 것을 고려 12-14.

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Formaldehyde</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">F-8775</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Glycine</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">G-8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Triton</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">T8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">35mm Glass bottom culture dishes</td>
			<td style="width:169px;">MatTeK corporation</td>
			<td style="width:187px;">P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Blocking solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92004 or DUO-92002</td>
			<td>provided in the Duolink PLA probes, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">VDAC1 antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:187px;">ab14734</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IP3R1-H80 antibody</td>
			<td style="width:169px;">Santa Cruz</td>
			<td style="width:187px;">sc28614</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CypD antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:187px;">ab110324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Grp75 antibody</td>
			<td style="width:169px;">Santa Cruz</td>
			<td style="width:187px;">sc13967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">TBS 10X</td>
			<td style="width:169px;">euromedex</td>
			<td style="width:187px;">ET220</td>
			<td>Dilute to obtain 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tween 100X</td>
			<td style="width:169px;">euromedex</td>
			<td style="width:187px;">2001-B</td>
			<td>dilute in TBS to obtain 0,01%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PLA Probes Mouse MINUS</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92004</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PLA Probes Rabbit PLUS</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92002</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Duolink detection reagents red</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ligation solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ligase</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Amplification solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Polymerase</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Duolink Mounting Medium</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO80102</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Softwares:</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Blob-finder software</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td>BlobFinder is a freely distributed software that can perform calculations on cells from fluorescence microscopy images. This software can be downloaded for free from The Centre for Image Analysis&#160;at Uppsala University who have developed the software and the work was supported by the EU FP6 Project&#160;ENLIGHT&#160;and&#160;&#160;Olink Bioscience. http://www.cb.uu.se/~amin/BlobFinder/index_files/Page430.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">ImageJ software</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td>Can be downloaded for free from: http://rsb.info.nih.gov/ij/download.html</td>
		</tr>
	</tbody>
</table>

study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

이 프로토콜을 사용하여 우리의 경험을 바탕으로, 우리는 안전하게 고정 된 세포에서 ER-미토콘드리아 상호 작용의 시각화 및 정량이 방법을 추천 할 수 있습니다. 항체의 여러 쌍을 사용 HuH7의 간암 세포주 인 시츄 근접 결찰 분석법 - 가시 ER-미토콘드리아 상호 작용의 대표 화상이 도시되어있다. 형광 현미경에 의해도 2에 도시 된 바와 같이, 각?...

Watch this Scientific Journal Video about Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells at JoVE.com

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

여기, 우리는 고정 된 세포에서 소포체와 미토콘드리아의 내생 적 상호 작용을 시각화하고 고감도로 정량화 할 수있는 절차를 설명합니다. 프로토콜은이 미토콘드리아 관련 막 계면에서 이노시톨에게 1,4,5- 트리 포스페이트 수용체 / 혈당 조절 단백질 75 / 전압 가변 음이온 채널 / 시클로 필린의 D 복합체 타겟팅 시츄 근접 결찰 분석법 최적화 기능.

소포체와 미토콘드리아의 상호 작용의 연구에 의해<em&gt; 현장에서</em&gt; 근접 내고 분석 고정 세포의

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

study-endoplasmic-reticulum-mitochondria-interactions-situ-proximity

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

24.5K 조회수.  Lund University.  여기, 우리는 고정 된 세포에서 소포체와 미토콘드리아의 내생 적 상호 작용을 시각화하고 고감도로 정량화 할 수있는 절차를 설명합니다. 프로토콜은이 미토콘드리아 관련 막 계면에서 이노시톨에게 1,4,5- 트리 포스페이트 수용체 / 혈당 조절 단백질 75 / 전압 가변 음이온 채널 / 시클로 필린의 D 복합체 타겟팅 시츄 근접 결찰 분석법 최적화 기?...

비디오: Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

효과기 - 단지에게 사용 BMP4의 다운 스트림 신호의 감지 현장에서 괞찮아, 근접 내고 분석

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

연구

생물학

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 ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

포유류의 세포에서 Endoplasmic 소포체 현지화 mRNAs의 시각화

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

유전자 발현의 DNA 상호 작용, 디스 전사 레귤레이터, 그리고 소설 안티 종양 에이전트를 식별 : 단백질을 연구하는 정량 분석

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

마이크로 RNA<em&gt; 현장에서</em&gt; 포르말린 고정 신장 조직에 대한 하이브리드

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

단일 미토콘드리아 과산화물 온전한 마음에 점멸 또는 공 촛점 이미징<em&gt; 생체</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

배양 된 세포의 소포체 하위 도메인의 시각화

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

정량적 RT-PCR에 의해 개별 인간 배아 줄기 세포를 프로파일 링

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

사용 소포체 칼슘 항상성 모니터링<em&gt; Gaussia</em&gt; 루시 페라 제 SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

소포체와 미토콘드리아의 상호 작용의 연구에 의해

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