We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

동물과 관련된 모든 절차는 노스 웨스턴 대학 기관 동물 관리 및 사용위원회 (IACUC)의 승인 지침에 따라 수행 하였다. 1. 마우스 모델 <ol><li> 운동 훈련에 8~12주 된 마우스를 사용합니다. 생체 내에서 행사에 의한 자식 작용을 감지하기 위해 영상 검사, 생화학 적 분석을위한 C57BL / 6 마우스의 GFP-LC3 형질 전환 마우스 (C57BL / 6 배경)를 사용합니다. </li></ol> 2. 운동에 의한 자식 작용 <ol><li> 러닝 머신 설치 - 강제 운동 <ol><li> 적응 및 2 일 동안 10 ° 오르막 오픈 러닝 머신에서 쥐를 훈련. 1 일에서 5 분 동안 마우스를 운동 8m / min으로하고, 2 일, 10m 또 다른 5 분 뒤에 8m / 분에서 5 분 동안 마우스를 운동 / 10 분. 부드러운 손을 찔러으로 실행하기 위해 쥐를 격려한다. </li><li> 3 번째 날에, 마우스는 10 ° 오르막 디딜 방아, starti에 90 분 동안 실행하는 하나의 한판 승부를 받아야 할10m / 분의 속도로 ng를. 40 분 후, 30 분의 총 1m / 분으로 10 매 분의 속도로 러닝 머신 속도를 증가시키고, 다음의 비율로 속도를 증가 1m /가 될 때까지 매 5 분 분 17m / 분 운동 시간 90 분 거리를 실행 1,070m의 총. </li><li> 낮은 강도 범위 (1.2 Hz에서)에 전기 자극을 설정하고 반복 발 충격을 방지하기 위해 실행하는 마우스 바랍니다. </li><li> 조직 컬렉션의 경우, 즉시 운동 후 자궁 전위 다음 이소 플루 란 흡입 쥐를 안락사. 기타 승인 안락사 방법은 또한 CO2 또는 다른 마취제 IP 주입이 사용될 수있다. 자식 작용의 수준은 안락사의 방법에 의해 영향을받는 것으로 보이지 않는다. </li></ol></li><li> 실행 휠 설정 - 자발적인 운동 <ol><li> 단일 보관 마우스로 케이지 안에 마우스 실행 휠 (11.4 cm 직경)을 놓습니다. </li><li> 자전거 주행 거리계를 사용하여 실행중인 용량을 확인합니다.358에서 [휠의 전체 회전 당 (mm)에 거리] 휠 매개 변수를 설정 (11.4 * π). 24 시간 후 주행 거리를 측정한다. </li><li> 마우스가 실행중인 휠 2 주 동안 자발적으로 실행할 수 있습니다. 조직 컬렉션의 경우, 실행 기간 후 아침에 마우스를 희생. </li></ol></li></ol> 3. 자식 작용 플럭스 평가 <ol><li> 휴식과 운동 조건에서 autophagic 플럭스를 측정하기 위해, 삼일의 자식 작용 억제제 클로로퀸과 쥐를 치료하고 PBS로 처리 된 마우스에 비교합니다. <ol><li> PBS (5 ㎎ / ㎖)에 클로로퀸을 용해하고, 복강 세 일 연속 50 ㎎ / ㎏ / 일의 용량으로 마우스로 클로로퀸을 주입. 쥐를 희생 마지막 주사 후 조직에게 3 시간을 수집합니다. </li><li> 운동 생쥐를 들어 50 ㎎ / ㎏ / 일의 투여 량으로 개의 연속 일 클로로퀸 그들을 전처리. 셋째 날, 후 클로로퀸의 동일한 농도로 마우스를 주입하고,90 분은 그들에게 또 다른 90 분 동안 러닝 머신에서 실행 할 수 있습니다. </li><li> 자궁 경부 전위 다음에 이소 플루 란 흡입 쥐를 안락사. 클로로퀸의 총 배양 시간 제어 (휴식) 마우스 (3 시간)과 동일 있도록 즉시 실행 한 후 조직을 수집합니다. 주 : 또는 마우스 조직에서 자식 작용 플럭스를 결정 클로로퀸의 단일 주입을 사용한다. </li></ol></li></ol> 4. 조직 수확 및 고정 <ol><li> 7.4의 최종 pH에서 PBS에서 : 조직 수확에 앞서 인산염 완충 생리 식염수 (PBS)와 신선한 4 % 파라 포름 알데히드 (필수 개인 보호 장비 PFA주의)을 준비합니다. 4 ° C에서 두 솔루션을 저장합니다. </li><li> (GFP-LC3 마우스 용) 4 % PFA 20 ㎖ - 15 30 ML의 주사기를 입력합니다. 20 G 카테터 바늘과 주사기를 연결합니다. </li><li> 운동 후, 자궁 경부 전위 다음에, 이소 플루 란 흡입하여 쥐를 안락사. </li><li> 그 바에서 깨끗한 작업 표면에 동물을 배치CK. 잘라 마음을 노출 다이어프램을 절단하여 흉강을 엽니 다. </li><li> 폐와 간은 완전히 창백 설정 될 때까지, 피가 나올 수 있도록 간장에 작은 절개를 확인하고 카테터를 통해 심장의 우심실로 천천히 15 ml의 4 % PFA의 총을 주입. / hr로 90 ㎖로 일정 유량 주사기 펌프를 이용하여 운동 세션 직후 마우스 관류. </li><li> 재관류 후, 바늘을 제거하고 관심 조직 (예 : 뇌 (11)과 골격근)를 수집합니다. 골격근의 경우, 광근 lateralis을 해부하다. 간략하게, 대퇴사 지역화 근육을 노출 뒤쪽 다리의 피부를 당겨 12 대퇴 및 대퇴골의 상부에 부착 된 외부 근육이다 광근 lateralis를 해부. </li><li> 또한 고정 및 탈수를 들어, 4 ° C에서 24 시간 동안 4 % PFA에 GFP-LC3 마우스의 조직을 배치 한 다음 15 %로 전송4 ° C에서 자당 PBS 하룻밤 또는 조직이 정착 될 때까지 다음 밤새 4 ° C에서 30 % 자당 PBS에 이상 저장. 그들은 강한 빛에 민감 할 수 있으므로 가능한 한 많은 어둠 속에서 샘플을 보관하십시오. </li><li> 웨스턴 블롯 분석을 위해 액체 질소에 직접 C57BL / 6 마우스에서 조직을 동결하고, -80 ° C에서 보관 웁니다. </li></ol> GFP-LC3 puncta의 5. 이미징 분석 <ol><li> 조직 프로세스 <ol><li> 같은 레이블이 작은 페트리 접시에 몇 분 동안 &quot;최적의 절단 온도 화합물&quot;OCT ()와 같은 매체를 포함에서 조직 (이전 PFA에 고정 30 % 자당에 저장)을 사전 처리합니다. </li><li> 조직을 충당하기에 충분한 신선한 OCT를 포함하는 표시 cryomold에 조직을 전송합니다. 방향을 cryomold의 아래쪽으로 절편 표면합니다 (광근의 크로스 섹션은 헤미 - 두뇌 근육과 시상 부분을 lateralis). 거품의 형성을 피할 것. </li><li> FREOCT를 흰색으로 바뀔 때까지, 드라이 아이스를 가득 덮여 거품 쿨러에 몇 분 동안 cryomold을 배치하여 샘플을 EZE. 레이블 포일에서 개별 샘플을 감싸 비닐 봉투에 밀봉하고,에서 -80 ° C 하룻밤 이상을 저장할 수 있습니다. </li><li> 슬라이드에 섹션을 10 ㎛의 두께로 샘플 부분을 절단하고, 마운트 그라 이오 스탯을 사용합니다. -20 ° C에서 슬라이드를 저장하고, 항상 가능한 빛으로부터 멀리 슬라이드를 유지합니다. </li></ol></li><li> 슬라이드 준비 <ol><li> 냉동기로부터 슬라이드를 제거하고 실온에서 이들을 해동. DAPI와 매체를 장착 한 방울 조직 절편을 커버. 상단에 적절한 크기의 커버 슬립을 놓고 거품 형성을 피하기. </li><li> , 커버 슬립의 가장자리를 밀봉 매니큐어가 실온에서 어둠 속에서 건조 할 수 있도록 다음 4 ° C에서 빛 밀폐 용기에 슬라이드를 저장하거나 GFP-LC3의 puncta의의 영상 캡쳐를 진행 매니큐어를 사용합니다. </li></ol></li><li> &lt;GFP의 강한&gt; 정량 - 형광 현미경에 의한 LC3의 puncta의 <ol><li> 모든 샘플에 대해 동일한 조건에서 사진 (노출 및 설정)을 캡처, 표면 형광 현미경을 수행합니다. GFP를 들어, 525 나노 미터의 방출 파장을 사용한다; DAPI를 들어, 490 nm의를 사용합니다. 광근 lateralis에 대한 60X 목적과 뇌의 전두엽 피질 영역을 사용하여 샘플 당 10 개 이상의 이미지를 획득. </li><li> 실험군 위해 멀게 통계 분석을 위해 각각의 이미지에서 2,500 ㎛의 (2)의 조직 영역에서의 GFP-LC3 puncta의 수를 정량화. &quot;객체 카운트&quot;자동 측정 기능을 이용하여, 각 샘플의 이미지 (10)의 평균 수를 계산한다. 광근에 대한 설정 근육이 lateralis : 임계 값 L = 10, H = 200; 배는 부드러운; 1X 깨끗하고 뇌의 전두엽 피질에 대한 설정은 다음과 같습니다 임계 값 L = 11, H = 200; 배는 부드러운; 1X 깨끗한. </li></ol></li></ol> 자식 작용 마커 6. 웨스턴 블롯 분석 <ol><li> &lt;강한&gt; 조직 프로세스 <ol><li> 용해 완충액 준비한다 : 50 mM 트리스 - 염산 단계; 150 mM의 NaCl을; 1 mM의 EDTA; 1 % 트리톤 X-100; 프로테아제 억제제 및 포스 파타 아제 억제제 (새로 추가). </li><li> C 스토리지 -80 °에서 C57BL / 6 마우스의 조직을 제거합니다. 다음 공정 전에 면도날 작은 조각으로 근육 조직을 잘라. (헤미 뇌에 대한) 800 μL 또는 샘플에 차가운 용해 버퍼 (광근 lateralis 용) 500 μl를 추가합니다. 중간 속도에서 균질화 4 ° C에서 샘플 균질화. </li><li> 완전 회전 4 ° C에서 1 시간에 다른 30 분 동안 균질 혼합물을 용균. </li><li> 4 ° C에서 10 분 동안 12,000 XG에서 균질를 스핀 다운 펠렛을 폐기하고 새로운 튜브에 뜨는를 수집합니다. </li></ol></li><li> 자식 작용 분석 <ol><li> 제조자의 규격에 따른 BCA 단백질 분석 키트를 사용하여 조직 용 해물의 단백질 농도를 결정한다. 각 샘플을 정상화동일한 농도. </li><li> 1에서 2 배 램 믈리 시료 완충액으로 샘플을 결합 : 1의 비율로, 5 ~ 95 ° C에서 끓인다 - 10 분, 및 P62 (항 P62 항체로, 자식 작용에 웨스턴 블롯 분석 (14), (13) 마커를 진행, 1 : 500 희석) 및 LC3 (항 LC3 항체, 1 : 500 희석). 얼음에 이상 배양이 LC3의 여러 성능이 저하 된 밴드를 생성 할 수 있으므로, 즉시 샘플 버퍼를 첨가 한 후 샘플을 끓인다. </li><li> ImageJ에 농도계를 사용하여 분석하여 P62 및 LC3 대역을 정량화하고, 대응 액틴 밴드 값을 정규화. </li></ol></li></ol>

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Immunology. 9, 324-337 (2009).</li><li>Miller, R. A., 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=Rapamycin-mediated+lifespan+increase+in+mice+is+dose+and+sex+dependent+and+metabolically+distinct+from+dietary+restriction.">Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction.</a> Aging Cell. 13, 10 (2014).</li><li>Grumati, P., 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=Physical+exercise+stimulates+autophagy+in+normal+skeletal+muscles+but+is+detrimental+for+collagen+VI-deficient+muscles.">Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles.</a> Autophagy. 7, 1415-1423 (2011).</li><li>Lira, V. 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The authors declare that they have no competing financial interests.

자식 작용은 에너지를 제공하고 세포질 구성 요소 나 손상된 세포 소기관의 리소좀 저하에 의해 세포 독성을 감소시키는 이화 과정이다. 자식 작용을 공부하는 것은 세포의 항상성의 조절 및 스트레스 반응의 메커니즘을 이해하는 것이 중요합니다. 새로운 모델과 방법론은 여러 병리학 적 과정 16, 17에 기여하는 방법을 손상 자식 작용 연구, 연구 분야 ...

자식 작용은 기아 저산소증 1, 2와 같은 다양한 스트레스 조건에 응답하여 유도 된 진화 적 보존 열화 경로이다. 자식 작용하는 동안 두 번 막 소포는, 불필요하거나 손상된 세포 내 구성 요소를 통합하고 분해 3 리소좀로 수송, autophagosomes을했다. 기저 자식 작용은 신경 퇴행, 종양 및 2 형 당뇨병 4, 5, 6을 포함하여 많은 질환에 연루되어 세포 기능 및 유기체 개발 및 손상된 기초 자식 작용을 위해 필수적이다. 가장 잘 알려진 생리 자식 작용의 유도는 기아이다. 그러나, 두 가지 제한 사항이 있습니다. 첫째, 기아는, 예를 들면, 효과적으로 동물에서 자식 작용을 유도하는 쥐의 음식 제한의 48 시간을 오랜 기간 소요대부분의 기관입니다. 둘째, 기아는 거의 때문에 뇌에서 상대적으로 안정적인 영양 공급, 뇌 자식 작용을 유도한다. 사실, 많은 약물이 혈액 뇌 장벽을 통과 할 수 없기 때문에, 저분자 유도제에 의해 자식 작용 유도를 검출하는 것이 곤란하다. 따라서, 더 좋은 질병 발병 자식 작용 활성의 기능을 분석하기 위해, 최근 운동 시간 7, 8, 9 단기간에 자식 작용을 유도하는보다 강력한 생리 방법임을 발견했다. 기아와 비교할 때, 자식 작용을 효과적으로 디딜 방아 빠른 30 분을 실행하여 유도된다. 따라서, 운동은 의료 혜택을 매개 질병을 예방 자식 작용의 메커니즘을 연구하는 편리하고 강력한 생리 학적 접근 방식이다. LC3 및 P62을 포함 자식 작용 활성의 검출을위한 여러 단백질 마커가있다. LC3 (미세 소관 - 관련 단백질 1A / 1B-빛 Chain 3) 세포질 단백질이다 (LC3-I는 자식 작용의 유도에 따라 PE (포스파티딜 에탄올 아민)에 결합된다)을 형성. PE-LC3 지질 화 (LC3-II 양식) autophagosomal 막에 채용하고 GFP로 표지 때 autophagosomes을 시각화 할 수있다. 현미경에서 autophagosomes의 구조를 반점하는 세포질에서의 전위는 자식 작용 유도의 표시입니다. P62 (예 유비퀴틴 단백질 등) 자식 작용 기판 용화물 수용체이고,도 autophagosomes에 포함된다. 단백질이 autophagosomes 함께 리소좀에서 분해되기 때문에, 그 농도는 자식 작용의 플럭스를 측정하는데 사용될 수있다. 여기에서 우리는 강제 운동 (러닝 머신)과 자발적 운동 (바퀴를 실행)을 포함한 유산소 운동에 의해 유발 된 다른 마우스 조직에서 자식 작용을 정량화하기 위해 이러한 마커를 사용하는 방법을 보여줍니다. 동일한 절차가 다른 유도제의 투여 후 생체 내 자식 작용의 측정에 적용 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Treadmill</td>
			<td style="width:204px;">Columbus Instruments</td>
			<td style="width:195px;">150-RM Exer 3/6</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mouse running wheel</td>
			<td style="width:204px;">Super Pet</td>
			<td style="width:195px;">100079365</td>
			<td>diameter 11.4 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Odometer</td>
			<td style="width:204px;">Bell</td>
			<td style="width:195px;">DASHBOARD 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe pump</td>
			<td style="width:204px;">KD Scientific</td>
			<td style="width:195px;">KDS100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fluorescence microscope</td>
			<td style="width:204px;">Nikon</td>
			<td style="width:195px;">Model: inverted microscope ECLIPSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cryostat</td>
			<td style="width:204px;">Leica</td>
			<td style="width:195px;">CM 1850UV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Homogenizer</td>
			<td style="width:204px;">IKA</td>
			<td style="width:195px;">003737001 / Model: T10 Basic S1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Chloroquine</td>
			<td style="width:204px;">CAYMAN CHEMICAL COMPANY</td>
			<td style="width:195px;">14194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Parafolmaldehye</td>
			<td style="width:204px;">SIGMA-ALDRICH</td>
			<td style="width:195px;">P6148</td>
			<td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mounting media</td>
			<td style="width:204px;">Vector Laboratories</td>
			<td style="width:195px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">p62 antibody</td>
			<td style="width:204px;">BD Biosciences</td>
			<td style="width:195px;">610833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LC3 antibody</td>
			<td style="width:204px;">Novus Biologicals</td>
			<td style="width:195px;">NB100-2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X Laemmli Sample Buffer</td>
			<td style="width:204px;">Bio-Rad Laboratories</td>
			<td style="width:195px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ImageJ</td>
			<td style="width:204px;">NIH</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

이 프로토콜은 유산소 운동으로 마우스 조직에서 자식 작용을 유도하는 두 가지 방법을 설명합니다 순응의 두 일까지 진행 다중 차선 러닝 머신에 강제 운동의 90 분의 총; 또는 단일 보관 마우스를 사용하는 실행중인 휠에 자발적 운동 2 주. 각각의 운동 프로토콜에서, 우리는 다양한 기관에서 형광 현미경 및 웨스턴 블롯 분석에 의한 자식 작용 플럭스를 측정 할 수 있습?...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

자식 작용의 활성화는 질병의 숫자의 예방에 유용하다. 생리 학적 접근 방법 중 하나는 생체 내에서 자식 작용은 운동이다 유도한다. 여기에서 우리는 유산소 운동으로 자식 작용을 활성화하고 마우스에서 자식 작용의 수준을 측정하는 방법을 보여줍니다.

마우스에 유산소 운동에 의해 자식 작용을 활성화

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

activating-autophagy-by-aerobic-exercise-in-mice

Research

JoVE Journal

Biology

11.7K 조회수.  Northwestern University.  자식 작용의 활성화는 질병의 숫자의 예방에 유용하다. 생리 학적 접근 방법 중 하나는 생체 내에서 자식 작용은 운동이다 유도한다. 여기에서 우리는 유산소 운동으로 자식 작용을 활성화하고 마우스에서 자식 작용의 수준을 측정하는 방법을 보여줍니다. 

비디오: Activating Autophagy by Aerobic Exercise in Mice

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

에어로빅 곰팡이에 의해 제어 플랜트 조직 분해에 대한 한천 - 블럭 Microcosms

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

연구

생물학

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

운동하는 동안 에너지 시스템의 공헌을 결정

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

마우스의 수력 학적 유전자 전달에 의한 단백질의 일시적 발현

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

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) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

자식 작용의 분석에<em&gt; 페니 chrysogenum</em&gt; 형광 현미경과 조합 기아 패드를 사용하여

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

젖 산 효소 격리 분석 결과-간단 하 고 신뢰할 수 있는 결정 하는 방법을 포유류 세포에서 대량 Autophagic 격리 활동

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

보 먼의 공간 2 광자 현미경에 의해 촉진 생쥐에서의 Micropuncture

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

마우스에 유산소 운동에 의해 자식 작용을 활성화

요약

더 많은 비디오 탐색

이 비디오의 챕터

에어로빅 곰팡이에 의해 제어 플랜트 조직 분해에 대한 한천 - 블럭 Microcosms

운동하는 동안 에너지 시스템의 공헌을 결정

마우스의 수력 학적 유전자 전달에 의한 단백질의 일시적 발현

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

자식 작용의 분석에

대변 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

젖 산 효소 격리 분석 결과-간단 하 고 신뢰할 수 있는 결정 하는 방법을 포유류 세포에서 대량 Autophagic 격리 활동

보 먼의 공간 2 광자 현미경에 의해 촉진 생쥐에서의 Micropuncture

마우스에 유산소 운동에 의해 자식 작용을 활성화

요약

더 많은 비디오 탐색

이 비디오의 챕터

에어로빅 곰팡이에 의해 제어 플랜트 조직 분해에 대한 한천 - 블럭 Microcosms

운동하는 동안 에너지 시스템의 공헌을 결정

마우스의 수력 학적 유전자 전달에 의한 단백질의 일시적 발현

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

자식 작용의 분석에

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

젖 산 효소 격리 분석 결과-간단 하 고 신뢰할 수 있는 결정 하는 방법을 포유류 세포에서 대량 Autophagic 격리 활동

보 먼의 공간 2 광자 현미경에 의해 촉진 생쥐에서의 Micropuncture

대변 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링