우리는 친절 pUTKan 식물 발현 벡터 구축 FRET 글루타민을 공급하기위한 플라스미드 박사 우베 Ludewig (호헨 하임 대학교) FRET 친절하게 포도당으로 우리를 제공하기 위해 교수 울프 프로 머 (과학 카네기 연구소, 스탠포드 대학) 감사합니다. AB는 BBSRC 대상 우선 순위 Studentships 프로그램에 의해 자금 지원됩니다. CK와 KP은 모두 바이오 처리에 RCUK 동호회 지원합니다. CK는 재정적 지원 론자 생물 의약품에 감사하고자합니다. 합성 생물학 및 혁신 센터는 관대 EPSRC에 의해 지원됩니다.

1. 세포주 부흥 및 유지 <ol><li> 8 mM의 L-글루타민, 10 ㎖ / L 100 배의 산틴 / 티미 딘 보충 (완전 성장 매체)로 보충 9 ML CD-CHO 매체에 CHO 세포를 부활. </li><li> 5 분 100 XG에 원심 분리기. </li><li> 신선한 완전 성장 배지 10 ㎖에 세포를 재현 탁. </li><li> 1 ㎖ 샘플을 제거하고 혈구에 트리 판 블루 염색 배제 방법을 사용하여 광학 현미경으로 가능한 세포 농도를 결정한다. </li><li> 125 ㎖의 3 × 10 5 세포 / ml의 파종 밀도 문화를 시작는 플라스크를 흔들어. </li><li> 120 rpm으로 회전하는 궤도 떨고 플랫폼에서, 37 ° C, 5 % CO 2의 가습 분위기 배양기에서 문화를 유지한다. </li><li> 2 × 105 세포 / ml의 접종 밀도로 완전한 성장 배지에서 계대마다 3-4일. </li></ol> 2. 플라스미드 C와 세포 라인의 형질바이오 센서 유전자를 ontaining <ol><li> 대규모 플라스미드 정제 키트를 사용하여 플라스미드 DNA를 준비한다. </li><li> (37 ° C, 5 % CO 2) 이전에 형질에 12 ~ 24 시간 세포가 적극적으로 형질 전환시 분할 보장하기 위해 적절한 조건에서 세포를 유지한다. </li><li> 트리 판 블루 염색 배제 방법을 사용하여 세포를 카운트 한 후 1 × 106 세포 / ml의 농도에서 진탕 플라스크 125 ㎖ 중의 세포 배양 물을 20 ㎖의 작동 부피를 준비한다. </li><li> 사용중인 세포주에 적합한 형질 전환 키트를 사용합니다. 형질 감염 시약 비율 플라스미드 DNA는 세포주 및 벡터에 의존 할 것이다. 그것은이가 전에 최종 형질을 실시하여 제조업체가 지정한 범위 내에서 비​​율의 범위를 테스트하여 최적화하는 것이 좋습니다. 또한 세포하지만 DNA를 포함하는 적어도 하나의 음성 대조군의 문화를 유지한다. </li><li> 정적 모드에서 4 일 동안 부화 후 SHAKI로 전송125 rpm으로 회전 NG 플랫폼입니다. </li><li> 킬 (kill) 곡선에 의해 결정되는 적절한 농도로 플라스미드 선택에 대한 적절한 항생제를 추가합니다. 본 연구에서는, 제 오신는 형질 세포의 선택을 위해 400 ㎍ / ㎖의 최종 농도로 첨가 하였다. </li><li> 제어 잘 세포가 사망 할 때까지, 항생제마다 추가 세포주에 대한 적절한 시간 간격으로 미디어를 변경합니다. </li><li> 문화를 동요로 진행하는 가장 합류 우물을 사용합니다. </li><li> 프리즈 믹스 1 ㎖에 10 7 가능한 세포를 포함하는 극저온 유리 병에서 세포를 동결 세포 은행을 설정합니다. 본 연구에서는이 92​​.5 % 성장 매체와 7.5 %의 디메틸 설폭 사이드로 구성되어 있습니다. </li></ol> 3. 배치 및 유가 세포 성장 곡선 <ol><li> 50 ㎖의 작업 볼륨 플라스크를 흔들어 250 ML의 형질 CHO 세포의 세중의 세포 배양을 설정하기 위해 위 세포의 유지 보수에 대한 지침을 따르십시오. </li><li> cultu 유지가습 셀 125 rpm으로 회전하는 궤도 떨고 플랫폼에 5 % CO 2와 37 ° C에서 인큐베이터,,, 24 시간 간격으로 각 성장 문화에서 4.1 ㎖의 시료를 제거있는 입술. </li><li> 트리 판 블루 염색 배제 방법으로 생존 세포의 농도와 세포 생존 능력을 확인하기 위해 샘플 100 μl를 사용합니다. </li><li> 가능한 세포 농도가 0으로 감소 될 때까지 반복합니다. </li><li> 유가 세포 배양이 각각 36 ㎜, 4 mm의 초기 값으로 자신의 농도를 복원하기 위해 6 일에 포도당 또는 글루타민의 적절한 양의 보충을위한 반복합니다. 그것은 순수의 동일 부피 (여기서는 12 ml)로 공급되어야하는 부가 제어 문화도 유지되어 추천. </li></ol> 4. 비율 측정을 FRET <ol><li> 4 ℃에서 5 분 100 XG에서 위의 단계 3.2 원심 분리기에서 분리 매일 시료 2 ㎖를 타고 </li><li> Resusp얼음 냉각 된 인산염 완충 식염수 (PBS)으로 2 ml의 세포 펠렛을 끝냈다. 6 잘 플레이트에이를 전송하고 잘 하나에 대한 세포를 포함하지 않는 빈 샘플을 추가합니다. </li><li> 즉시 435분의 465 NM (파란색)과 510분의 520 nm의 (노란색)의 435분의 430 nm의 방출 파장의 여기 파장에 파란색과 노란색 형광의 수준을 측정한다. </li><li> FRET 비율 (검출 파란색 형광 검출에 노란색 형광의 비율)을 계산합니다. </li></ol> 5. 대사 산물 분석 실험 <ol><li> 4 ℃에서 5 분 100 XG에서 위의 단계 3.2 원심 분리기에서 분리 매일 시료 2 ㎖를 타고 뜨는을 제거합니다. </li><li> 5 분 100 XG에 다시 3 ㎖의 얼음 차가운 PBS와 원심 분리기에 세포 펠렛을 Resuspend. </li><li> 뜨는을 제거하고 위 3 ㎖ 얼음 차가운 PBS에 세포 펠렛을 resuspend. 15 초 펄스에서 3 분마다 15 초 오프 샘플 배를 초음파 처리. 이때, 셀 여분원하는 경우 CTS는 고정 할 수 있습니다. </li><li> (자료 표 참조) 세포 내 포도당 농도를 결정하기 위해 세포 추출물의 샘플에 대한 제조 업체의 지침에 따라 적절한 혈당 분석 키트를 사용합니다. 참고 : 표준 곡선을 구성하는 적절한 기준을 사용합니다. 본 연구에서는 표준의 농도는 0 ~ 100 밀리미터 사이였다. </li><li> 표준에 대한 측정 값을 사용하여 표준 곡선을 생성하고 (이 경우 : Y = 1310.51x - y는 흡광도 읽기와 x는 포도당 농도입니다 723.43) 최적의 선형 방정식을 찾을 수 있습니다. </li><li> 표준 곡선을 사용하여 계정에 희석 복용 포도당 농도를 계산한다. 세포 내 포도당 농도는이 식을 이용하여 계산 될 수있다 : <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> CHO 세포의 경우, 단일 셀 볼륨은 12 μM (12)의 기포 지름을 이용하여 산출했다 </SUP&gt;. </li><li> (자료 표 참조) 세포 내 글루타민 농도를 결정하기 위해 세포 추출물의 샘플에 대한 제조 업체의 지침에 따라 적절한 글루타민 분석 키트를 사용합니다. 참고 : 표준 곡선을 구성하는 적절한 기준을 사용합니다. 본 연구에서는 표준의 농도는 0-2 사이 밀리미터였다. </li><li> 상기 5.5에서와 같이, (Y는 흡광도 판독 및 x는 Y = + 17.1 203.1x는 글루코스 농도가이 경우에) 검정 곡선을 작도 </li><li> 표준 곡선을 사용하여 계정에 희석을 고려하여 샘플의 글루타민 농도를 계산한다. 세포 내 글루타민 농도는 상기 수학 식 1을 사용하여 계산 될 수있다. </li></ol>

<ol>
	<li>Moser, I., Jobst, G., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor+arrays+for+simultaneous+measurement+of+glucose,+lactate,+glutamate,+and+glutamine.">Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine.</a> Biosens. Bioelectron. 17 (4), 297-302 (2002).</li><li>Billingsley, K., 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=Fluorescent+nano-optodes+for+glucose+detection.">Fluorescent nano-optodes for glucose detection.</a> Anal. Chem. 82 (9), 3707-3713 (2010).</li><li>Dattelbaum, J. D., Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Determination+of+Glutamine+Using+a+Genetically+Engineered+Protein.">Optical Determination of Glutamine Using a Genetically Engineered Protein.</a> Anal. Biochem. 291 (1), 88-95 (2001).</li><li>Kfouri, M., 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=Toward+a+miniaturized+wireless+fluorescence-based+diagnostic+imaging+system.">Toward a miniaturized wireless fluorescence-based diagnostic imaging system.</a> IEEE J. Sel. Top. Quantum Electron. 14 (1), 226-234 (2008).</li><li>Dittrich, P. S., Manz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+detection+in+microfluidic+channels&#8212;the+Holy+Grail+in+muTAS.">Single-molecule fluorescence detection in microfluidic channels&#8212;the Holy Grail in muTAS.</a> Analytical and Bioanalytical Chemistry. 382 (8), 1771-1782 (2005).</li><li>Hou, B. H., Codamo, J., Pilbrough, W., Hughes, B., Gray, P. P., Munro, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sensors+for+monitoring+dynamic+changes+of+intracellular+metabolite+levels+in+mammalian+cells.">Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells.</a> Nat. Protoc. 6 (7), 1818-1833 (2011).</li><li>Bermejo, C., Haerizadeh, F., Takanaga, H., Chermak, D., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+cytosolic+glucose+and+ATP+levels+in+yeast+using+optical+sensors.">Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.</a> Biochem. J. 432, 399-406 (2010).</li><li>Yang, H. Y., Bogner, M., Stierhof, Y. D., Ludewig, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H(+)-independent+glutamine+transport+in+plant+root+tips.">H(+)-independent glutamine transport in plant root tips.</a> Plos One. 5, (2010).</li><li>Fehr, M., Takanaga, H., Ehrhardt, D. W., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+high-cavacity+bidirectional+glucose+transport+across+the+endoplasmic+reticulum+membrane+by+genetically+encoded+fluorescence+resonance+energy+transfer+nanosensors.">Evidence for high-cavacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors.</a> Mol. Cell. Biol. 25 (24), 11102-11112 (2005).</li><li>Ai, H. W., Hazelwood, K. L., Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET+pairs+for+ratiometric+imaging+of+dual+biosensors.">Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors.</a> Nat. Methods. 5 (5), 401-403 (2008).</li><li>Ouyang, M. X., Sun, J., Chien, S., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+hierarchical+relationship+of+Src+and+Rac+at+subcellular+locations+with+FRET+biosensors.">Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (38), 14353-14358 (2008).</li><li>Han, 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=Cultivation+of+recombinant+Chinese+hamster+ovary+cells+grown+as+suspended+aggregates+in+stirred+vessels.">Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.</a> J. Biosci. Bioeng. 102 (5), 430-435 (2006).</li><li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+optimization+of+a+family+of+genetically+encoded+metabolite+sensors+by+semirational+protein+engineering.">Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering.</a> Protein Sci. 14 (9), 2304-2314 (2005).</li><li>Wong, D. C. F., Wong, N. S. C., Goh, J. S. Y., May, L. M., Yap, M. G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+N-Glycosylation+gene+expression+in+CHO+cell+Fed-batch+cultures.">Profiling of N-Glycosylation gene expression in CHO cell Fed-batch cultures.</a> Biotechnol. Bioeng. 107 (2), 516-528 (2010).</li><li>Behjousiar, A., Kontoravdi, C., Polizzi, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Monitoring+of+Intracellular+Glucose+and+Glutamine+in+CHO+Cell+Culture.">In Situ Monitoring of Intracellular Glucose and Glutamine in CHO Cell Culture.</a> PLoS One. 7, (2012).</li><li>Dwyer, M. A., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periplasmic+binding+proteins:+a+versatile+superfamily+for+protein+engineering.">Periplasmic binding proteins: a versatile superfamily for protein engineering.</a> Curr. Opin. Struct. Biol. 14 (4), 495-504 (2004).</li></ol>

저자가 공개하는 게 없다.

FIBS는 방법을 담금질 추출에서 발생하는 불확실성을 제거,이 경우 성장을 제한 영양소로, 생체 내 및 주요 분자의 현장 정량에 수 있습니다. 결과는 FIBS 신호 및 글루타민 및 글루코스 0.3-2 mm를 위해 1-5 mm 인 세포 내 농도 간의 상관 관계가 있다는 것을 시사한다. 배치 CHO 세포 배양,이 농도는, 지수 정지, 초기 하락 단계에서 발생된다. 지수 및 정지상은 적절한 공급 전략 디자인의 관?...

대사 산물의 모니터링은 공정 개발, 미디어 및 공급 설계 및 대사 공학을 포함한 바이오의 다양한 응용 프로그램을 가지고 있습니다. 다양한 방법은 효소 1, 화학 2, 3 또는 결합 분석을 사용하여 농도를 측정 가능하다. 흥미로운 옵션은 형광등을 사용하는 것입니다, 단백질 기반의 주요 대사 산물의 세포 내 농도의 기자로 생체 내 생물학적 센서에. 소형화는 실제로 신호 대 잡음비의 4,5을 향상시키고 단백질 기반 센서 유전자 제공된 외인성 시약 대사 분석 필요 없다는 의미를 인코딩 할 수있는 매우 낮은 볼륨 내 분석, 형광 편리한 도구이다. 포스터 공명 에너지 전달 (FRET) 바이오 센서는 리간드 결합 도메인에 의해 연결된 두 개의 형광 단백질로 구성되어 있습니다. FRET은 수용체의 형광 분자 locat입니다에 사진 흥분 기증자로부터의 에너지의 비 복사 전송합니다<html> 근접 (&lt;100)에 에드. 리간드 결합 또는 벽개 차례로, 발광 스펙트럼의 변화에​​ 의해 측정 FRET 효율의 변화로 이어지는, 형광체의 근접성의 변화를 유도하고, 센서의 형태 적 변화를 일으키는. FRET 집적 생물학적 센서 (FIBS)는 구성 적 세포주 내에서 생산되고, 그 스펙트럼 특성은 세포 내의 대사 물질의 농도에 따라 변경. FIBS 동적 모델 6에 대한 측정을위한 이상적 빠른 응답 시간이있다. 이전 응용 프로그램은 7-9 단일 및 다중 열 대사를 모니터링하고 시공간 분포 (11)에 데이터를 제공하는 것을 포함. 리간드 결합 에너지 전송 및 리간드 결합 가까이 접촉 (그림 1)에 두 개의 형광 물질을 제공 아포 민을 낮추는 형광의 근접을 방해 아포 - 최대 : FIBS는 두 가지 구성으로 만들 수 있습니다. 이 작품에서 _content은 &quot;&gt;, 프로토콜은 구조적으로 신진 대사, 포도당 또는 글루타민 FIBS를 표현 (CHO) 세포주 중국어 햄스터 난소 구축을 위해 제공됩니다. 방법은 생체 내에서 센서의 교정에 설립되어 미래의 정량을 사용하도록 도 2에 제시된 것처럼. 이것을 기초 세포 내 대사 산물 농도의 측정은, 글루코스 또는 글루타민의 세포 내 농도가 어느이 영양분이 공정 중 높은 농도로 첨가하는, 유가 식 CHO 세포 배양에서 결정될 수있다. 결과 그것은 비 침습적, 저가이므로 독립적 효소 분석에 의해 확인 된 FIBS 및 이전 구성 검량선으로부터 형광 신호를 이용하여, 세포 내 농도의 정확한 예측이 가능하다는 것을 보여준다.이 방법은 현재의 분석 기술을 통해 상당한 이점을 제공한다 및 패스트 월 될 수 FIBS의 실시간 신호를주는문화 전반에 걸쳐 itored.

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		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
</table>

fibs-enabled noninvasive metabolic profiling

전산 생물학의 시대에, 새로운 높은 처리량 실험 시스템은 그들이 예측 목적의 유효성을 검사 할 수 있도록 모델을 채우고 세분화하기 위해 필요합니다. 이상적으로 그러한 시스템은 타임 코스 데이터가 수득 될 때 샘플링 및 파괴 분석을 배제 저용량, 것이다. 필요한 것은 실시간으로 그리고 간접적으로 필요한 정보를보고 할 수있는 시츄 모니터링 도구이다. 흥미로운 옵션은 형광등을 사용하는 것입니다, 단백질 기반의 세포 내 농도의 기자로 생체 내 생물학적 센서에. 대사 산물의 정량에 응용 프로그램을 발견했습니다 생체 바이오 센서의 하나의 특정 클래스는 리간드 결합 도메인에 의해 연결된 두 개의 형광 단백질 사이의 포스터 공명 에너지 전달 (FRET)을 기반으로합니다. 통합 생물 센서 (FIBS를) FRET이 구조적으로 세포주 내에서 생산, 그들은 빠른 응답 시간과 자신의 스펙트럼 차이세포 내 대사 산물의 농도에 따라 변경 racteristics. 본 논문에서는 세포 내 대사 산물 농도의 미래 정량을 가능하게하기 위해 구조적으로 포도당과 글루타민 FIBS를 표현 (CHO) 세포주 중국어 햄스터 난소를 구성하고 배치 세포 배양 생체 내에서 FIBS를 교정하는 방법이 설명되어 있습니다. 유가 식 CHO 세포 배양에서의 데이터 FIBS가 세포 내 농도의 결과 변화를 검출하는 각각의 경우에 수 있었다고 보여준다. 독립적 효소 분석에 의해 확인 된 FIBS 및 이전 구성 검량선으로부터 형광 신호를 이용하여, 세포 내 농도를 정확하게 측정 하였다.

방법론의 개요는 그림 2에 표시됩니다. 여기에 제시 한 연구에서, CHO 세포 FIBS 벡터와 안정 세포주에 형질 전환 된 400 ㎍ / ㎖의 오신의 항생제 압력에 선정되었다. 구조적으로 포도당과 글루타민 센서를 발현 개의 분리 안정한 세포주 따라서 생성되었다.도 1은 본 연구에서 사용 된 두 개의 바이오 센서의 구성을 도시한다. 글루코스 센서는 아포 - 맥스 원리에 근거하고 대...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

현장 신진 대사 프로파일 링을위한 포스터 공명 에너지 전달을 통합 생물 센서 (FIBS)를 보정하는 방법에 대한 설명이 표시됩니다. FIBS는 비 침습적 메타 모델과 생물 공정 조건의 높은 처리량 검사의 발달을 돕는 대사 산물의 세포 내 수준을 측정하는데 사용될 수있다.

비 침습 대사 프로파일 링에게 FIBS 지원

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

fibs-enabled-noninvasive-metabolic-profiling

Research

JoVE Journal

Bioengineering

9.6K 조회수.  Imperial College London.  현장 신진 대사 프로파일 링을위한 포스터 공명 에너지 전달을 통합 생물 센서 (FIBS)를 보정하는 방법에 대한 설명이 표시됩니다. FIBS는 비 침습적 메타 모델과 생물 공정 조건의 높은 처리량 검사의 발달을 돕는 대사 산물의 세포 내 수준을 측정하는데 사용될 수있다. 

비디오: FIBS-enabled Noninvasive Metabolic Profiling

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

높은 처리량 MicroRNA의 프로파일 : 최적 멀티 플렉스 플루 동적 ArrayTM의 IFCs에 Nanoliter의 규모 qRT - PCR

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

연구

생체공학

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

현장 압축 하중과 뼈, 치주 인대 치아 섬유 공동의 상관적인 비 침습 이미징

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

정량화 및 조정 가능한 저항 펄스 감지 기능을 사용하여 세포 외 소포의 크기 프로파일 링

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Micropost 배열에 쉽고 정확한 - 기계 프로파일

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

N을 사용 하 여 Sialic Acid의 대사 Glycoengineering-acyl-Mannosamines를 수정

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

대사 지원 Excised, 자기 공명 현미경 중 생활 뇌 조직

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry:에 대 한 강력 하 고, 비 침 투 적인 접근 세포 에너지 대사를 조사

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors 프로 테아 제 활동에 감지 비보에 비 침 투 적인 진단에 대 한

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

글로벌 세포 매트릭스 Metalloproteinase 및 3D Hydrogels 신진 대사 활동 측정

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

매트릭스 세포 대사 크로스토크 연구를 위한 인간 3D 세포외 세포외 매트릭스-세포세포 배양 모델

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...