Name: measuring trans-plasma membrane electron transport by c2c12 myotubes
Uploaded: 2018-05-04T15:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

우리는 그들의 기술 지원에 대 한 토마스 벨, 린 Mattathil, 마크 Mannino, 및 Neej 파 텔에 게 감사 하 고 싶습니다. 이 작품은 조나단 피셔 국립 연구소의 당뇨병과 소화기 및 신장 질환 (NIDDK)에서 미국 공중 보건 봉사 상 R15DK102122에 의해 지원 되었다. 원고 내용은 전적으로 저자의 책임 이며 반드시는 NIDDK 또는 국립 보건원의 공식 의견을 대표 하지 않는다.

참고:에 대 한 주요 단계 도식 개요 그림 1 을 참조 하십시오.
1. 서 부 표준시-1 감소 분석 결과
<ol><li>성장 하 고 96 잘 접시 행 A-f.를 활용 하 여 표준 셀 문화 절차7 을 사용 하 여 C2C12 부착 세포 분화<ol>
<li>구성 된 Dulbecco의 수정이 글의 중간 (DMEM)의 하 2% 말 혈 청, 100 U/mL 페니실린과 0.1 mg/mL 스 보충 차별화 매체를 사용 합니다. 5% CO2와 37 ° C에서 세포를 품 어.</li>
		<li>TPMET에서 하는데 참여 모니터링, 100 µ M ascorbic 산 차별화 미디어 보충. 셀 ~ 24-48 대 한 차별화 미디어에는 알을 품을 수 h.</li>
	</ol>
</li>
	<li>재고 서 부 표준시-1 및 PMS 솔루션을 준비 합니다.<ol>
<li>서 부 표준시-1의 10 밀리미터 주식을 하려면 분해 서 부 표준시-1의 0.033 g (수식 무게 (FW): 651.34 g/mol) 5 ml의 인산 염 버퍼 식 염 수 (PBS). 4 ° c.에 게</li>
		<li>PMS의 5 밀리미터 주식을 하려면 0.0023 g PMS의 분해 (FW: 306.34 g/mol) 이온된 H2O의 1.5 ml (diH2O). -20 ° C에서 저장 하 고 빛 으로부터 보호 합니다.</li>
	</ol>
</li>
	<li>11.4 ml PBS 또는 HEPES 5mm의 최종 농도 식 염 수 버퍼 0.0108 g 포도 당의 추가 (HBS; 20 mM HEPES 나트륨 소금, 140 m NaCl, KCl, 2.5 m m MgSO4, 5mm 1 m CaCl2m m). 10 m m 400 µ M의 최종 농도를 48 µ L 5mm 20 µ M의 최종 농도 대 한 PMS의 서 부 표준시-1의 480 µ L를 추가 합니다.</li>
	<li>TPMET에서 하는데 참여, 모니터링 솔루션 두 6 mL aliquots 나눕니다. 한 약 수에 diH2O 6 µ L을 추가 하 고 다른 약 수 2 구/mL AO 2 U/mL의 최종 농도 6 µ L 추가 합니다.</li>
	<li>TPMET에 superoxide 참여 모니터링 솔루션 두 6 mL aliquots 나눕니다. 한 약 수를 0.1 M KPO4 버퍼의 55 µ L을 추가 하 고 다른 약 수를 60 U/mL의 최종 농도 대 한 6.5 구/mL 잔디의 55 µ L을 추가 합니다.</li>
	<li>PBS로 세포 세척. 미디어를 발음 하 고 150 µ L PBS를 추가 합니다. 다음에 PBS 발음. 참고: 분석 결과 도금, 연결 된 셀과 함께 이루어집니다. 따라서, 원심 분리 또는 세척 과정에서 trypsin의 사용 이다.</li>
	<li>서 부 표준시-1 솔루션 (-)의 100 µ L를 추가 잔디 또는 (-) 아오 열 1-6를 100 µ L의 서 부 표준시-1 솔루션 (+) 잔디 또는 (+) 아오 열 7-12 96 잘 접시에 추가. 배경 컨트롤 행 G와 H를 사용 하 여 (즉, 변화를 모니터링 하는 데 셀 없이 웰 스에서 혼자 시 흡 광도에).</li>
	<li>438에서 1 시간 마다 10 분 분 광 광도 계를 사용 하 여 흡 광도 값 nm.</li>
	<li>읽기 후에, 미디어를 발음 하 고 각각을 씻어 PBS의 150 µ L로 잘. 다음에 PBS 발음.</li>
	<li>그 잘 시간 언제 든 지 흡 광도에서 잘에 대 한 초기 흡 광도 빼서 각 잘 흡 광도 변화를 계산 합니다. 흡 광도 (있을 경우) 배경 웰 스 (즉, 분석 결과 솔루션 우물만 셀)에서 관찰 된 변화에 대 한이 변경 수정.</li>
	<li>분석을 위해 60 분 제어 측정에 흡 광도 데이터 정상화 또는 PBS와 HBS 웰 스를 세척 하 고 bicinchoninic 산 (BCA) 단백질 분석을 수행.</li>
	<li>2 mg/mL 소 혈 청 알 부 민 (BSA) 기준 추가 (0.5-2에서 셀의 합류의 정도 따라 표준 곡선 µ L) 빈 우물 (행 G 및 H), 다음 추가 BCA 시 모든 우물에.</li>
	<li>단백질의 µ g 당 서 부 표준시-1 감소의 nmol 통해 데이터를 계량. 멸종 계수로 37 m m-1 c m-1 22 를 사용 하 여 감소 된 서 부 표준시-438에서 1 nm.</li>
</ol>2. DPIP 감소 분석 결과
<ol><li>성장 하 고 C2C12 단계 1.1 동일한 절차와 부착 세포를 차별화 합니다.</li>
	<li>다음과 같이 재고 10 m m DPIP 솔루션을 준비 합니다. 분해 DPIP의 0.029 g (FW: 290.08 g/mol) diH2O. 확인 600에서 흡 광도 측정 하 여 DPIP의 농도의 10 mL에 nm는 분 광 광도 계. 소 광 계수로 1 m m-1 c m-1 23 을 사용 하 여 감소 DPIP 600에 대 한 nm. 4 ° c.에 게</li>
	<li>5mm의 최종 농도 대 한 PBS의 11.880 ml 0.0108 g의 포도 당을 추가 합니다. 100 µ M의 최종 농도 대 한 10 m m DPIP의 120 µ L를 추가 합니다.</li>
	<li>TPMET에서 하는데 참여, 모니터링 솔루션 두 6 mL aliquots 나눕니다. 한 약 수에 diH2O 6 µ L을 추가 하 고 다른 약 수 2 구/mL AO 2 U/mL의 최종 농도 6 µ L 추가 합니다.</li>
	<li>TPMET에 superoxide 참여 모니터링 솔루션 두 6 mL aliquots 나눕니다. 한 약 수를 0.1 M KPO4 버퍼의 55 µ L을 추가 하 고 다른 약 수를 60 U/mL의 최종 농도 대 한 6.5 구/mL 잔디의 55 µ L을 추가 합니다.</li>
	<li>미디어를 발음 하 고 PBS의 150 µ L에서 세포를 씻어. PBS를 발음 하 고 100 µ L DPIP 솔루션 (-)의 추가 잔디 또는 (-) 아오 열 1-6를 100 µ L DPIP 솔루션 (+) 잔디의 또는 (+) 아오 열 7-12 96 잘 접시에 추가. 배경 컨트롤 행 G와 H 사용 (즉, 변화를 모니터링 하는 데 셀 없이 웰 스에서 혼자 시 흡 광도에).</li>
	<li>사용 하 여 600 nm에서 흡 광도 측정 분 광 광도 계 10 분 마다 1 헤에 대 한 계량 60 분 단계 1.9 1.13 비슷한 컨트롤에 상대적인 흡 광도 있는 변화.</li>
</ol>3. 결정 여부 감소 전자 수락자는 AO 또는 잔디에 대 한 기판의
<ol><li>PBS의 HBS 5.436 ml, 10 m m 400 µ M의 최종 농도 5mm 20 µ M의 최종 농도 대 한 PMS의 24 µ L에 대 한 서 부 표준시-1 240 µ L를 추가 합니다.<ol>
<li>DPIP, PBS의 HBS 5.940 mL를 100 µ M의 최종 농도 10 m m, DPIP의 60 µ L를 추가 합니다.</li>
	</ol>
</li>
	<li>각 셀의 부재에서 평면 아래 96 잘 접시에 잘 438에서 분 광 광도 계에서 흡 광도 측정 하는 솔루션의 100 µ L를 추가 nm, DPIP에 대 한 서 부 표준시-1, 또는 600 nm에 대 한.</li>
	<li>100 μ M의 최종 농도 대 한 웰 스의 절반의 10mm 하는데 1 µ L를 추가 합니다. 안정화 될 때까지 흡수도 모니터링 합니다.</li>
	<li>안정화, 시 각 음을 2 U/mL의 최종 농도 대 한 200 U/mL AO의 1 µ L를 추가 하거나 각 60 U/mL의 최종 농도 잘 하 고 1 시간에 대 한 흡 광도 모니터 6 구/mL 잔디의 1 µ L를 추가 합니다.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microtiter+plate+assay+for+superoxide+dismutase+using+a+water-soluble+tetrazolium+salt+(WST-1).">A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1).</a> Clinica Chimica Acta. 293 (1-2), 157-166 (2000).</li><li>Higaki, Y., 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=Oxidative+stress+stimulates+skeletal+muscle+glucose+uptake+through+a+phosphatidylinositol+3-kinase-dependent+pathway.">Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway.</a> Am J Physiol Endocrinol Metab. 294 (E889-E897), (2008).</li><li>Zuagg, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+characteristics+and+some+chemical+propertiesof+N-methylphenazinium+methyl+sulfate+(phenazine+methosulfate)+and+pyocyanine+at+the+oxidation+level.">Spectroscopic characteristics and some chemical propertiesof N-methylphenazinium methyl sulfate (phenazine methosulfate) and pyocyanine at the oxidation level.</a> J Biol Chem. 239 (11), 3964-3970 (1964).</li><li>Dayan, J., Dawson, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+specificity+of+ascorbate+oxidase.">Substrate specificity of ascorbate oxidase.</a> Biochem Biophys Res Commun. 73 (2), 451-458 (1976).</li><li>Bellavite, P., della Bianca, V., Serra, M. C., Papini, E., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+of+neurtrophils+forms+superoxide+anion+but+does+not+reduce+cytochrome+c+and+dichlorophenolindophenol.">NADPH oxidase of neurtrophils forms superoxide anion but does not reduce cytochrome c and dichlorophenolindophenol.</a> FEBS. 170 (1), 157-161 (1984).</li><li>Berridge, M. V., Tan, A. S., McCoy, K. D., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemical+and+cellular+basis+of+cell+proliferation+assays+that+use+tetrazolium+salts.">The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts.</a> Biochemica. 4, 15-20 (1996).</li><li>Tan, A. S., Berridge, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+produced+by+activated+neutrophils+efficiently+reduces+the+tetrazolium+salt,+WST-1+to+produce+a+soluble+formazan:+a+simple+colorimetric+assay+for+measuring+respiratory+burst+activation+and+for+screening+anti-inflammatory+agents.">Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents.</a> J Immunol Methods. 238 (1-2), 59-68 (2000).</li><li>Phillips, P. 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=Myricetin+induces+pancreatic+cancer+cell+death+via+the+induction+of+apoptosis+and+inhibition+of+the+phosphatidylinositol+3-kinase+(PI3K)+signaling+pathway.">Myricetin induces pancreatic cancer cell death via the induction of apoptosis and inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway.</a> Cancer Letters. 308 (2), 181-188 (2011).</li><li>Altundag, E., 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=Quercetin-induced+cell+death+in+human+papillary+thyroid+cancer+(B-CPAP)+cells.">Quercetin-induced cell death in human papillary thyroid cancer (B-CPAP) cells.</a> Journal of thyroid research. 2016 (8), (2015).</li><li>Ukeda, H., Kawana, D., Maeda, S., Sawamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Assay+for+Superoxide+Dismutase+Based+on+the+Reduction+of+Highly+Water-soluble+Tetrazolium+Salts+by+Xanthine-Xanthine+Oxidase.">Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction of Highly Water-soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase.</a> Biosci Biotechnol Biochem. 63 (3), 485-488 (1999).</li><li>Halaka, F. G., Babcock, G. T., Dye, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+5-methylphenazinium+methyl+sulfate.+Reaction+of+the+oxidized+form+with+NADH+and+of+the+reduced+form+with+oxygen.">Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen.</a> J Biol Chem. 257 (3), 1458-1461 (1982).</li><li>Maghzal, G. J., Krause, K. H., Stocker, R., Jaquet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+species+derived+from+the+family+of+NOX+NADPH+oxidases.">Detection of reactive oxygen species derived from the family of NOX NADPH oxidases.</a> Free Radic Biol Med. 53 (10), 1903-1918 (2012).</li></ol>

저자는 공개 없다.

우리는 세포 외 전자 수락자, DPIP, 및 서 부 표준시-1 tPMET C2C12 myotubes에서 모니터링 하는 spectrophotometric 분석 실험에 활용 하는 두 가지 방법 제시. 표준 문화 절차에 셀 라인의 성장과 플레이트 리더를 분 광 광도 계 분석 결과 간단한 microplate에에서 이러한 전자 수락자와 tPMET 모니터링 가능 하다. 서 부 표준시-1 감소는 잘-투-잘 분석 결과, 내에서 재현할 수 이지만 일상적인 변화. PBS는 버퍼를 활용 ...

전자 수락자를 줄이기 위해 순화 된 플라즈마 막 수 플라즈마 멤브레인 고유의 redox 용량1는 보기를 주도하 고 있다. 이전에 곰 팡이, 식물, 그리고 동물에서 본, tPMET는 여러 생물2,3,,45에 공통 프로세스입니다. 특히,이 과정은 Saccharomyces cerevisiae, 당근 세포, 적혈구, 림프 톨, 다리, 흑색 종, 대 식 세포, 골격 근육, 및 호 중구2,3, 에서 입증 되었습니다. 4 , 5 , 6 , 7. extracellular oxidants를 줄이기 위해 플라즈마 막에 걸쳐 전자를 수송 하는 과정에서 tPMET는 포함 하는 많은 세포 기능에 관여: 세포 성장5,8, 세포 생존 능력9, 철 대사10,11,,1213, 반응성 산소 종12,,1415보호를 신호 하는 세포입니다. 많은 세포질 기능에 tPMET의 개입으로 인해 tPMET의 불균형은 암16,17, 심혈 관 질환 포함 한 몇 가지 심각한 건강 상태의 발전에 기여할 가설 되었습니다 및 대사 증후군18.
원형질 막의 전자 전송을 모니터링 하는 여러 가지 하지만 가장 널리 사용 되는 기술 색도계 분석 실험을 통해 세포 외 전자 수락자의 감소를 평가 하는 것입니다. 일반적으로 사용 되는 세포 외 전자 수락자 tetrazolium 소금, DPIP, FeCN, 그리고 ferricytochrome c19,20있습니다. 가장 일반적으로 사용 되는 tetrazolium 소금은 2 세대 소금 알려진 서 부 표준시-119. 이 화합물은 1 세대 tetrazolium 소금 두 된 그룹,21의 물 가용성 증가 하는 때문에 비해 색도계 분석 실험에 활용 하기가 쉽습니다. 중간 전자 수락자 1-methoxy-phenazine methosulfate (Mpm)와 함께에서 서 부 표준시-1, 2 단 전자 전송 이벤트에서 감소 된다. 이 감소는 더 강렬한, 노란색 formazan20,22에 약하게 색 산화 형태의 서 부 표준시-1을 변경합니다. 서 부 표준시-1 높은 분석 결과 감도21,22로 이어지는 37 x 103 M-1c m-1의 높은 어 금 니 소 광 계수가 있다. DPIP는 또한 tPMET를 모니터링 하는 extracellular 전자 수락자로 이용 된다. 그것은 그 DPIP 감소 될 수 있다 extracellularly 중간 전자 수락자23,24의 도움 없이 tPMET에 의해 표시 되었습니다. 중간 전자 수락자의 부족, DPIP 픽업 전자를 서 부 표준시-124달리 플라즈마 막에서 직접 수 있습니다. DPIP와 마찬가지로 FeCN 줄어들 extracellularly ferrocyanide 중간 전자 수락자19,24의 도움 없이 tPMET에 의해에 표시 되었습니다. 서 부 표준시-1, DPIP와 달리 FeCN 낮은 분석 결과 감도9로 이어지는 낮은 어 금 니 소멸 계수가 있다. TPMET 모니터를 또 다른 일반적으로 사용 되는 세포 외 전자 수락자는 ferricytochrome c. 서 부 표준시-1, ferricytochrome c 감소 증가 중간 전자 수락자, Mpm22의 사용과 비슷합니다. 서 부 표준시-1과는 달리, ferricytochrome c 방법은 높은 배경 및 낮은 분자량 소멸 계수22덜 민감한.
여기 우리 spectrophotometric 분석 실험을 통해 tPMET의 실시간 분석을 위한 방법 제시. 메서드 활용 서 부 표준시-1 및 DPIP, extracellular 전자 수락자로 그들은 둘 다 높은 어 금 니 소멸 계수 되 고 일반적으로 다른에 비해 덜 비싼 사용 ferricytochrome c 등 세포 외 전자 수락자. 우리는 phenazine methosulfate (PMS) 활용 Mpm 대신 그들은 유사한 화학 메이크업 및 PMS 있다 훨씬 덜 비싼. Mpm은 photochemically 안정 긴 수명이 필요로 하는 상용 키트에 대 한 중요 한 특성입니다. 그러나, 우리가 있도록 PMS 신선한 각 분석 결과 대 한 안정성 문제 해서는 안됩니다. 우리는 또한 가능한 효소 사이 상호 작용 ( 그림 1참조) extracellular 전자 수락자와 더 tPMET의 과정을 특성화 하기 위해 활용 될 수 있습니다 효소를 평가 하는 방법을 제시. 특히, 효소 AO와 잔디를 사용할 수 있습니다 어떤의 tPMET 부분은 하는데 전송 또는 extracellular superoxide 릴리스, 원형질 막에 걸쳐 수송 될 전자에 대 한 두 가지 일반적인 방법 결정 합니다.

<table><tbody><tr><td>C2C12 myoblasts</td><td>American Type Culture Collection&amp;nbsp;</td><td>CRL-1772</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium - low glucose</td><td>Sigma</td><td>D6046</td><td></td></tr><tr><td>Fetal Plex animal serum complex</td><td>Gemini Bio-Products&amp;nbsp;</td><td>100-602</td><td></td></tr><tr><td>penicillin-streptomycin</td><td>Sigma</td><td>516106</td><td></td></tr><tr><td>horse serum</td><td>Gibco Technologies</td><td>16050-130</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>15 cm culture dishes</td><td>TPP</td><td>93150</td><td></td></tr><tr><td>96 well culture plates</td><td>TPP</td><td>92096</td><td></td></tr><tr><td>2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1)</td><td>Accela ChemBio&amp;nbsp; Inc</td><td>SY016315</td><td></td></tr><tr><td>phenazine methosulfate&amp;nbsp;</td><td>Sigma</td><td>P9625</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>ascorbate oxidase&amp;nbsp;</td><td>Sigma</td><td>A0157</td><td></td></tr><tr><td>superoxide dismutase&amp;nbsp;</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>2,6-dichloroindophenol sodium salt&amp;nbsp;</td><td>ICN Biomedicals</td><td>215011825</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>HEPES sodium salt</td><td>Sigma</td><td>H3784</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>potassium chloride</td><td>Fisher Scientific&amp;nbsp;</td><td>BP366</td><td></td></tr><tr><td>magnesium sulfate heptahydrate</td><td>Sigma</td><td>M5921</td><td></td></tr><tr><td>calcium chloride dihydrate</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>potassium phosphate</td><td>Fisher Scientific&amp;nbsp;</td><td>BP363</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Powerwave X-I spectrophotometer</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr><tr><td>Spectronic Genesys 5 Spectrophotometer</td><td>Thermo Scientific</td><td>336001</td><td></td></tr><tr><td>PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterile</td><td>MidSci</td><td>781602</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma</td><td>220299</td><td></td></tr><tr><td>Iron (II) sulfate heptahydrate</td><td>Sigma</td><td>215422</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma</td><td>H9636</td><td></td></tr><tr><td>xanthine oxidase</td><td>Sigma</td><td>X4500</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>R Studio</td><td>Rstudio</td><td></td><td>https://www.rstudio.com/products/rstudio/</td></tr><tr><td>KC4</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr></tbody></table>

measuring trans-plasma membrane electron transport by c2c12 myotubes

Trans 플라스마 막 전자 전송 (tPMET) extracellular oxidants에 의해 세포내 감소 스트레스 로부터 세포의 보호 뿐만 아니라 손상 으로부터 보호 역할을 하고있다. Extracellular oxidants 세포내 reductants에서 전자 수송의이 과정은 잘 정의 된. 여기 우리 C2C12 myotubes tPMET 세포 외 전자 수락자를 활용 하 여 모니터링 하 여 spectrophotometric 분석 실험을 제시: 수용 성 tetrazolium 소금-1 (서 부 표준시-1)과 2, 6-dichlorophenolindophenol DPIP (DCIP). 이러한 전자 수락자의 감소, 통해 우리는 실시간 분석에이 과정을 모니터 할 수 있습니다. 효소 superoxide dismutase (SOD)는 분석 실험을 하는데 산화 효소 (AO) 등의 추가 함께 우리 tPMET의 부분 각각 하는데 수출 또는 초과 생산 때문입니다 확인할 수 있습니다. 서 부 표준시-1 낮은 배경 가진 안정적인 결과를 표시 했다, 그러나 DPIP AO와 spectrophotometric 분석 시연 했다 잔디의 추가 후 다시 산화 될 수 있었다. 이 메서드는 실시간, 빠른 멀티 잘 spectrophotometric 분석 결과 tPMET, ferricyanide (FeCN) ferricytochrome c 감소 등을 모니터링 하는 데 사용 하는 다른 방법을 통해 장점 보여 줍니다.

통계는 RStudio 통계 소프트웨어25를 사용 하 여 반복된 측정 ANOVA와 수행 했다. 샘플 크기는 그림 범례에 표시 됩니다.
모니터 tPMET, C2C12 myotubes extracellular 전자 수락자, 서 부 표준시-1 및 DPIP 함께 활용 했다. AO는 서 부 표준시-1의 부분을 결정 하는 데 사용 되었다, DPIP 감소 하는데 경과 예정 이었습니다 그리고 ?...

Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

C2C12 Myotubes에 의해 트랜스-플라즈마 막 전자 전송 측정

이 프로토콜의 목표 spectrophotometrically 트랜스-플라즈마 막 전자 전송 extracellular 전자 수락자를 활용 하 여 모니터링 하 고 이러한 세포 외 전자 수락자로 발생할 수 있는 효소의 상호 작용을 분석 하는입니다.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

measuring-trans-plasma-membrane-electron-transport-by-c2c12-myotubes

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

6.9K 조회수.  Saint Louis University. 이 프로토콜의 목표 spectrophotometrically 트랜스-플라즈마 막 전자 전송 extracellular 전자 수락자를 활용 하 여 모니터링 하 고 이러한 세포 외 전자 수락자로 발생할 수 있는 효소의 상호 작용을 분석 하는입니다.

비디오: Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아 격리 사용

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

연구

발생학

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

이식 근육 전구 세포를 타겟팅 할 수 분자 이미징

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

의학

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

소우의 자손에서 이전에 냉동 된 심장 조직에서 미토콘드리아 전자 전송 복합체 측정 : 운동 유발 미토콘드리아 생물 에너지 변화를 평가하는 모델

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

생화학

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Na+에 대한 내부 미토콘드리아 막 민감도, 부분적으로 분할된 기능적 CoQ 풀 공개

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

경피적 바늘 생검에 의해 획득 골격근 조직에서 고립 된 미토콘드리아의 준비 및 호흡계 평가

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

C2C12 Myoblast 세포를 사용하여 Bioenergetic 프로필 실험

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

생물학

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

C2C12 근육 모세포의 세포에 근긴장 성 이영양증 1 모델링

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

미토콘드리아 절연 마우스 중심부에 투과율 전환 기공 영업의 다중 매개 변수 측정

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...