우리는 도움과 이러한 프로토콜을 설정하는 조언 박사 부부 칼 - 하인츠 크라우스와 빈센트 JAQUET에 감사하고, 또한 자신의 기술 지원 NCCR의 바이오 이미징 플랫폼 및 박사 나빈 Gopaldass에서 크리스토프 바우어 제롬 Bosset 감사합니다. 연구는 스위스 국립 과학 재단 (National Science Foundation)의 ProDoc의 교부금에 의해 지원됩니다.

1. 시각화 및 phagosomes를있는 ROS 생산의 질적 측정 OBG - 코팅 된 비드를 미리 준비한다. 비즈 코팅 절차 및 한천 오버레이 기술은 출판 참조 5,14에서 적응됩니다. <ol><li> 1.5 ML 튜브에 3.0 μm의 카르 실리카 비드 (약 1.8 × 10 9 구슬) 1 ㎖ 현탁액을 추가, 비즈 빠른 스핀과 소용돌이로 교반하여 1 ㎖의 PBS로 3 배 씻는다. </li><li> 25 ㎎ / ㎖ 시안 아미드를 (공유 BSA를 prelabeled 바인딩 카르 실리카 비드를 활성화하는 방법)를 포함하는 PBS 1 ㎖에 구슬을 재현 탁, 15 분 동안 바퀴에 품어. </li><li> 커플 링 버퍼의 1 ㎖로 세척 배 빠른 스핀으로 (0.1 M 붕산 나트륨, 산도 8.0) (또는 5 초 동안 테이블 탑 미니 원심 분리기의 최대 속도로 원심 분리기 또는 테이블 톱 원심 분리기에서 1 분 2,000 XG에 원심 분리기)와 과량의 시안 아미드를 제거하는 텍싱. </li><li> C 500 μL로 세척 구슬을 혼합OxyBurst 그린 (OBG) H2HFF (디 하이드로-2 &#39;, 4,5,6,7,7&#39;- hexafluorofluorescein)-BSA의 1 ㎎을 함유하는 버퍼를 oupling 표준 상한까지 고로 가스 수에서 질소 가스 튜브를 채우고, 어둠 속에서 실온에서 14 시간 (야간)의 바퀴에 품어. </li><li> 구슬이 (PBS에서 글리신 250 ㎜)의 빠른 스핀과 소용돌이로 교반하여 담금질 버퍼를 제거하는 커플 링 버퍼로 두 번 반응 OBG를 제거하고 버퍼를 급냉 1 ㎖로 2 배 씻는다. </li><li> BSA에 활용하는 알렉사 형석 594 숙신 에스테르 50 μg을 함유하는 커플 링 버퍼의 1 ML을 추가합니다. 모자를 씌우기 전에 질소 주입 튜브, 그리고 어둠 속에서 실온에서 1.5 시간 동안 바퀴에 품어. </li><li> 구슬이 빠른 스핀과 소용돌이로 교반하여 1 ㎖의 PBS로 3 배 세척 한 후, 구슬이 빠른 스핀과 반응을 중지 소용돌이로 교반하여 버퍼를 급냉 1 ㎖로 3 배 씻으십시오. </li><li> 마지막으로, 장기 저장을위한 V 지드 / w 10 %의 2 μL로 PBS 1 ㎖에 비드를 재현 탁. concentratio을 측정혈구 (보통 1 ~ 2 × 10 9 구슬 / ㎖)와 서스펜션에있는 구슬의 N은, 모자를 씌우는 전에 질소 튜브를 채우고, 어둠 속에서 4 ° C에 보관하십시오. </li><li> OBG의 형광의 코팅 효율은 500 nm에서 여기를 이용한 방출 스펙트럼을 확인하여 시험 될 수있다. 양 고추 냉이 퍼 옥시 다제 (HRP)의 존재하에 H 2 O 2에 의해 산화되면, 발광 피크 (538 NM)에서 산화 비드의 형광 강도는 nonoxidized 코팅 비드에 비해 11 ~ 12 배 높다. </li><li> 수확은 기하 급수적으로 광학적으로 투명한 플라스틱이나 유리 바닥에 3cm 접시에 세포의 밀도가 다른 접시, 밤새 그들을 성장, 10cm 페트리 접시에서 Dictyostelium의 세포를 성장. 실험에 대해 80 %의 합류 요리를 선택합니다. Dictyostelium의 세포 배양에 대한 자세한 프로토콜 15을 발표했습니다. </li><li> 하세요! loflo 매체 (LF 매체)로 배지를 교체, 알을 품다HL5C 배지의 세포 및 intraendosomal 형광도를 감소시키기 위하여 실험 전 2 시간. </li><li> 병행 10-15 기다려, 약 1 ㎜ 두께 한천 층을 형성하기 위해 평탄면 (x 10cm 10cm의 즉 유리판)에 한천을 넣어 LF 배지에 10 ml의 1.5 % 박토 한천 녹지 분 응고한다. 나중에 사용하기 위해 LF 매체에 2cm X 2cm 사각형과 장소에 한천 층을 잘라. </li><li> 한편, 회전하는 디스크 또는 넓은 필드 현미경을 준비하고 22 ° C의 환경 챔버의 온도를 설정하고이 실험에 대한 설정을 조정합니다. (토론에 추가 의견을 참조하십시오.) </li><li> 배양 2 시간 후, 3cm 접시로부터 LF 매체를 대기음하지만 세포 단일 층은 여전히​​ 매체의 박막에 의해 보호되어야한다. 1.5 × 10 7 구슬 / ㎖로 코팅 구슬을 희석하고, 세포 층에 10 μl를 추가합니다. </li><li> 하나 스퀘어 한천 장을 가지고, 과잉 액체를 배출하지만 계속 젖은 상태. 조심스럽게 일을 넣어전지 층의 위에 전자 한천 사각형. 그것은 세포에 누워 때 한천 광장을 이동하지 마십시오. 한천 오버레이함으로써 흡수를 향상 구슬과 세포 사이의 접촉을 증가시키는 데 사용하고, 또한 약간의 대물 초점면에 더 잘 유지, 세포를 압축한다. </li><li> , 접시에 뚜껑을 배치 현미경 스테이지에 배치하고 자동으로 적색, 녹색 및 위상 채널에서 사진을 찍고 2 시간 이상마다 1 분. </li><li> 선택 식균 작용의 전 과정을 포함하는 세포 사건에 초점을 맞 춥니 다. 최적화 된 3 채널 병합 및 전문 화상 처리 소프트웨어를 사용하여 동영상으로 사진을 조립한다. 빨간색과 녹색 채널에서 선택한 각 구슬의 형광 강도를 정량화하고, 그린 / 레드의 비율은 세포의 동적 phagosomal ROS 생산을 반영합니다. </li></ol> 2. 세포 내 과산화물 생산의 정량 및 중간 처리량 측정 <ol><li> (을)를 수집NE 80 %의 합류 HL5C 배지 10 ㎖에 Dictyostelium의의 요리. 5 신중 분, 완전히 대기음 초과 매체 850 XG에 원심 분리기 Dictyostelium의 세포. SS6.4 버퍼에 재현 탁 세포 [소렌슨 버퍼에 0.12 M 소르비톨 (14.69 밀리미터 KH 2 PO 4, 6.27 mM의 나 2 HPO 4), pH를 6.4], 셀 카운트 및 6 × 106 세포 / ml의 최종 농도로 희석 (토론에 추가 의견을 참조). </li><li> (토론에 추가 의견을 참조) 흰색 불투명 한 96 웰 플레이트의 각 웰에 세포 현탁액의 50 μl를 추가합니다. </li><li> 필요한 경우 각이 잘 멀티 채널 피펫을 사용하여에 DHE 재고 (DMSO 30 ㎜)로 희석 DHE의 SS6.4, 피펫 50 μl의 500 배 희석. DHE의 최종 농도는 30 μM이다. 반응이 즉시 시작됩니다. </li><li> 자극 또는 억제 특정 요구에 따라이 시점에서 추가, 또는 다른 시간 지점에서 할 수있다. </li><li> 엔드 포인트 &quot;최고 읽기 &amp; #을 사용하여(34) 522 nm/605 nm에서 형광 여기 / 방출과 형광 마이크로 플레이트 리더의 모드는, 22 ℃에서 1 시간, 중간 흔들림 5 초 / 분마다 2 분을 읽어 </li></ol> 3. 양적 및 세포 외의 높은 처리량 측정 H 2 O 2 생산 <ol><li> 2.1 및 2.2과 동일한 단계를 따릅니다. </li><li> SS6.4, 소용돌이 10 ㎖에 HRP 주식의 5 μL (증류수 100 U / ㎖)를 첨가하여 희석 한 HRP를 준비하거나 잘 혼합하고 나중에 사용하기 위해 얼음에 계속 튜브를 반전. 희석 된 HRP 솔루션은 0.05 U / ㎖에 있습니다. </li><li> 각각 AUR 재고 (DMSO 10 ㎜ AUR)와 6.25 μM의 최종 농도에 SS6.4 버퍼에 희석 한 HRP 솔루션, 0.005 U / ㎖를 혼합하여 AUR 반응 혼합물을 준비합니다. 예로서, 40 반응에, 반응 혼합물을 제조 희석 HRP의 400 μL와 AUR 스톡 용액 2.5 μL로 SS6.4 1600 μL를 혼합한다. </li><li> AUR 믹스의 50 μl를 추가합니다필요한 경우 각이 잘 멀티 채널 피펫을 사용하여에 진짜야. 지금 반응이 시작됩니다. </li><li> 자극 또는 억제 특정 요구에 따라이 시점에서 추가, 또는 다른 시간 지점에서 할 수있다. </li><li> 530 nm/590 nm에서 형광 여기 / 방출과 형광 마이크로 플레이트 리더의 종점 &quot;최고 읽기&quot;모드를 사용하여, 22 ℃에서 1 시간, 중간 흔들림 5 초 / 분마다 2 분을 읽어 </li></ol>

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관심 없음 충돌 선언하지 않습니다.

전술 한 방법에 비해, OBG 분석법 동적 대신 세포 집단에서 보통 ROS 신호 측정, 단일 세포 수준에서 phagosomal ROS 생성의 과정을 시각화한다. 이러한 인구 평균 방법은 비동기 식균 작용에 의한 중요 정보를 모호하게하는 경향이있다. 우리는 성공적으로 Dictyostelium의에 DHE와 AUR 분석을 적용하고 프로토콜을 최적화. 가장 중요한 것은, 우리는이 두 가지 분석에 의해 측정 된 유형과 ROS의 세포 내 ?...

활성 산소 종 (ROS)는 같은 호스트 방어, 신호, 조직 개발 및 부상에 대한 응답뿐만 아니라 고혈압과 암과 같은 생물 학적 과정의 다양한 참여하고 있습니다. 잘 공부 phagosomal의 NADPH 산화 효소 기계는 호중구 &#39;phagosomes를 1 섭취 박테리아를 죽이고, 산화 버스트로 알려진 신속한 ROS 생성에 최선을 다하고 있습니다. 또한, 미토콘드리아 호흡 사슬의 부산물로서 전자의 누출은 이전에는 ROS의 규제 소스에 대한 책임을 져야하는 것으로 생각되었다. 그러나 최근에는 마우스 대 식세포 2 박테리아의 intraphagosomal 살인에 기여하는 중요한 메커니즘으로 확인되었다. 최근 몇 년 동안, 사회 아메바, Dictyostelium의는 타고난 면역 반응의 세포 고유의 메커니즘을 연구 할 수있는 강력하고 인기있는 모델이되고 있습니다. 실제로, Dictyostelium의 인간 식세포는 molecula 보전의 놀라 울 정도로 높은 수준을 공유박테리아, 말림을 감지하고 3,4 살인에 대한 책임을 R의 기계. 단백질과 같은 NADPH 산화 효소, 카탈라제, 슈퍼 옥사이드 dismutases으로 ROS의 생산 해독 관련 효소의 동족체는, 인간과 Dictyostelium의 모두에서 찾을 수 있습니다. 최고의 공부 아메바으로, Dictyostelium의 포유 동물 모델 시스템에 대한 몇 가지 독특한 장점을 제공한다. 그들은 8-10 시간의 배가 시간으로, CO 2에 대한 필요없이 상온에서 성장한다. 그들은 쉽게 부착 또는 현탁액 문화로 유지 될 수있다. 또한, 자신의 완전한 염기 서열과 주석 반수체 게놈, 쉬운 유전자 조작 덕분에, Dictyostelium의 매우 매력적인 실험 모델 생물이되고있다. 이전의 연구에서, ROS에 의한 산화 후 형광을 방출 (이하 OxyBurst 녹색, OBG라고도 함) 형광의 다양한 염소와 불소 유도체, ROS의 기자로 사용되어왔다. 세포 투과 동부 표준시휴대 impermeant 덱스 트란 또는 단백질 결합 유도체는 ROS 세포를 측정하기 위해 사용되는 반면 erified 유도체, 세포질 ROS를 측정하는 데 사용된다. 특히, OBG BSA 코팅 된 비드 이미 포유류 세포 소재 phagosomal ROS의 생성을 검출하기 위해 사용되어왔다. 그러나, 마이크로 플레이트 리더 기반 접근법은 단지 세포 집단에서 평균화 ROS 생성 커브를 줄 수있다. 본 프로토콜로, Dictyostelium의 전문 식세포, 최적화 실험 조건을 사용하여, 우리는 구슬에 어떤 opsonin 공역없이 안정적이고 효율적으로 식균 작용을 구하십시오. DHE는 ROS 생산 6-9를 검출하는 그러한 포유류 호중구 및 대 식세포 등 다양한 모델 시스템에 사용되어왔다. 한편, 방법 8,10의 민감도와 특이도를 통해 몇 가지 논쟁이있다. Amplex 레드, AUR을 측정하는 것이 더 적합하다 산도에 덜 민감의 형광 강도의 개선 된 버전으로nonneutral 또는 약산성 milieus에서 ROS. AUR은 최근 여러 포유류의 시스템 (11, 12)에 적용되어 있지만, 꽤 자주 약산성 성장 미디어를 필요로 nonmammalian 모델에서의 사용은 아직보고되지 않았습니다. 또한, 정량적 동적 사회적 아메바 Dictyostelium의에서 ROS 생산 및 국산화를 측정 할 수있는 게시 된 프로토콜이 없습니다. 프로토콜 제시된 세트의 목표는 다양한 ROS 및 현지화를 모니터링하기 쉽고 다양한 솔루션을 제공하고, 또한 ROS 관련 세포 메커니즘에 대한 통찰력을 제공한다. OBG 분석을 위해, 우리는 Dictyostelium의 세포로 OBG 코팅 구슬의 흡수 후 phagosomal의 ROS 생성의 전 과정을 모니터링하는 라이브 현미경을 사용하고, 박테리아의 intraphagosomal 살인의 메커니즘을 연구하기 위해 새로운 접근 방식을 제공했다. 우리는 세포 내 superox을 측정하기 위해, Dictyostelium의 매체 처리량 DHE와 AUR 분석를 최적화강력한 ROS 자극으로 LPS를 사용하여 각각 IDE 및 세포 H 2 O 2 생산. LPS 최근 포식 균 (13)를 향해 Dictyostelium의의 살균 활성을 증가 표시했다합니다. 또한, DEDTC 및 catalase의 치료는 명시 적으로 두 가지 방법이 구체적으로 다양한 종류와 Dictyostelium의에서 ROS의 세포 내 현지화를 측정하는 것을 확인했다. 마지막으로, OBG 분석은 상기 동물 세포의 식세포 수용체에 대한 리간드와 함께 비즈 opsonizing 의해, 어떤 접착 식세포 세포에 적용 할 수있다. 원칙적으로, DHE와 AUR 분석은 미세 조정 적절한 실험 조건에서 어떤 부착 또는 비 접착 세포에서 측정 할 수있다.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>OxyBURST Green H2HFF BSA</td>
			<td>Invitrogen</td>
			<td>O-13291</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-20004</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylated silica beads</td>
			<td>Kisker Biotech</td>
			<td>PSi-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>HL5C medium</td>
			<td>ForMedium</td>
			<td>HLC0102</td>
			<td></td>
		</tr>
		<tr>
			<td>LoFlo medium</td>
			<td>ForMedium</td>
			<td>LF1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>204010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium</td>
			<td>Sigma</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed</td>
			<td>Invitrogen</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Roche</td>
			<td>10108090001</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyldithiocarbamate (DEDTC)</td>
			<td>Sigma</td>
			<td>D3506-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C9322-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanamide</td>
			<td>Sigma</td>
			<td>187364-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L2630-25G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>ibidi &#956;-Dish 35 mm, high</td>
			<td>ibidi</td>
			<td>81156</td>
			<td>Bottom made of optically clear plastic</td>
		</tr>
		<tr>
			<td>MatTek dish 35 mm</td>
			<td>MatTek corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Bottom made of a glass coverslip</td>
		</tr>
		<tr>
			<td>Scepter Cell Counter</td>
			<td>Merck Millipore</td>
			<td>PHCC00000</td>
			<td></td>
		</tr>
		<tr>
			<td>White 96 well plate</td>
			<td>Nunc</td>
			<td>236108</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SORVALL</td>
			<td>Legend RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy Mx</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system

활성 산소 종 (ROS)는 동적으로 상호 전환 또는 하나 촉매 또는 자발적으로 제거하는 반응과 짧은 수명, 산소 함유 분자의 범위를 포함한다. 때문에 대부분의 ROS와 그 소스와 세포 내 현지화의 다양성의 짧은 제품 수명에 완전한 그림은 프로토콜의 조합을 사용하여주의 측정에 의해 얻어 질 수있다. 여기에서, 우리는 OxyBurst 그린 (OBG)를 사용하여 세 가지 다른 프로토콜의 집합 코팅 구슬, 또는 dihydroethidium (DHE)를 제시하고 Amplex UltraRed (AUR)는 질적 및 양적 같은 Dictyostelium의 등 전문 식세포의 다양한 ROS를 모니터링 할 수 있습니다. 우리는 동적으로 단일 세포 수준에서 intraphagosomal ROS의 생성을 시각화하는 비즈 코팅 절차 및 사용 OBG 코팅 구슬과 라이브 현미경을 최적화. 우리는 E.에서 지질 다당류 (LPS)를 확인 Dictyostelium의에서 ROS의 생성을위한 자극제로 대장균. 또한, 우리는 거라고eveloped 실시간 정량적으로 각각 세포 내 과산화물 및 세포 H 2 O 2의 생산을 측정하기 위해 DHE와 AUR를 사용하여 중간 처리량 분석.

된 phagosomes에 ROS의 생성은 현미경 (파일 S1)에 의해 성적으로 동적으로 시각화 할 수 있습니다. 알렉사 형석 594에 의해 방출되는 적색 형광은 pH를 구분하고 OBG의 산화 녹색 채널의 형광을 증가하는 동안, phagosomal 환경에서 일정하게 유지된다. 체외의 방출 스펙트럼은 산화 nonoxidized OBG 코팅 비드 산화 후의 강도의 상당한 증가를 나타내는,도 1b에 비교된다. 라이브 이...

Watch this Scientific Journal Video about Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System at JoVE.com

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

우리는 정량적 연구를위한 각종 아메바 및 포유류의 세포 모델에 적용 할 수있는 활성 산소 종 (ROS)의 측정을위한 일련의 프로토콜을 채택.

검출 시각화 및 아메바 모델 시스템에서 활성 산소 종의 생성을 정량

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

detecting-visualizing-quantitating-generation-reactive-oxygen-species

Research

JoVE Journal

Biology

16.0K 조회수.  University of Geneva.  우리는 정량적 연구를위한 각종 아메바 및 포유류의 세포 모델에 적용 할 수있는 활성 산소 종 (ROS)의 측정을위한 일련의 프로토콜을 채택. 

비디오: Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

에 잔 심장을 떠올리 Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

연구

생물학

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

전자 스핀 공명 산소 매핑에 대한 라이브 종의 마이크로 이미징

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

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 ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

셀 침습적 DISC1 단백질 종의 생성, 정제 및 특성

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

모델 시아 노 박테리아 종의 표시 및 Markerless 돌연변이의 생성

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

심근 성능 및 산소 소비의 연구에 대한 설치류 작업 심장 모델

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

탐식의 초기 단계를 시각화

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

활성 산소 종의 전환 가능한 protoporphyrin IX 의존성 광생성에 의한 마우스 피부의 줄기 세포 틈새 및 조직 재생 자극 in situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...