この研究は、東ポーランド2007-2013(POPW.01.03.00-06-003/09-00)、ルブリンのジョン・ポール2世カトリック大学(イロナ・サドクのための若い科学者助成金)のバイオテクノロジーと環境科学の学部によって、東ポーランドの運用プログラム開発の下で欧州地域開発基金から欧州連合によって支援されました OPUS13(2017/25/B/NZ4/01198マグダレーナ・スタニシェフスカ、原則調査官)。 著者らは、細胞培養およびタンパク質定量のための装置を共有してくれたJPII CULの学際研究センター、酸化ストレス研究所のアニエスカ・シシビオール教授に感謝する。

<ol>
	<li>Li, F., Zhang, R., Li, S., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IDO1:+an+import+ant+immunotherapy+target+in+cancer+treatment.">IDO1: an import ant immunotherapy target in cancer treatment.</a> International Immunopharmacology. 47, 70-77 (2017).</li><li>Heng, B., 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=Understanding+the+role+of+the+kynurenine+pathway+in+human+breast+cancer+immunobiology.">Understanding the role of the kynurenine pathway in human breast cancer immunobiology.</a> Oncotarget. 7 (6), 6506-6520 (2015).</li><li>Badawy, A. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kynurenine+pathway+and+human+systems.">Kynurenine pathway and human systems.</a> Experimental Gerontology. 129, (2020).</li><li>Chen, Y., Guillemin, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kynurenine+pathway+metabolites+in+humans:+Disease+and+healthy+states.">Kynurenine pathway metabolites in humans: Disease and healthy states.</a> International Journal of Tryptophan Research. 2 (1), 1-19 (2009).</li><li>Sathyasaikumar, K. V., 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=Xanthurenic+Acid+Formation+from+3-Hydroxykynurenine+in+the+Mammalian+Brain:+Neurochemical+Characterization+and+Physiological+Effects.">Xanthurenic Acid Formation from 3-Hydroxykynurenine in the Mammalian Brain: Neurochemical Characterization and Physiological Effects.</a> Neuroscience. 367, 85-97 (2017).</li><li>Malina, H., Richter, C., Frueh, B., Hess, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+epithelial+cell+apoptosis+and+intracellular+Ca2++increasein+the+presence+of+xanthurenic+acid.">Lens epithelial cell apoptosis and intracellular Ca2+ increasein the presence of xanthurenic acid.</a> BMC Ophthalmology. 2, 1-7 (2002).</li><li>Col&#237;n-Gonz&#225;lez, A. L., Maldonado, P. D., Santamar&#237;a, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Hydroxykynurenine:+an+intriguing+molecule+exerting+dual+actions+in+the+central+nervous+system.">3-Hydroxykynurenine: an intriguing molecule exerting dual actions in the central nervous system.</a> Neurotoxicology. 34, 189-204 (2013).</li><li>Xue, P., Fu, J., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aryl+hydrocarbon+receptor+and+tumor+immunity.">The aryl hydrocarbon receptor and tumor immunity.</a> Frontiers in Immunology. 9 (286), 00286 (2018).</li><li>Tan, V. X., Guillemin, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kynurenine+pathway+metabolites+as+biomarkers+for+amyotrophic+lateral+sclerosis.">Kynurenine pathway metabolites as biomarkers for amyotrophic lateral sclerosis.</a> Frontiers in Microbiology. 13 (1013), 1-11 (2019).</li><li>Wilding, J. L., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+for+drug+discovery+and+development.">Cancer cell lines for drug discovery and development.</a> Cancer Research. 14 (40), 2377-2384 (2015).</li><li>Takikawa, O., Kuroiwa, T., Yamazaki, F., Kido, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+interferon-gamma+action.+Characterization+of+indoleamine+2,3-dioxygenase+in+cultured+human+cells+induced+by+interferon-gamma+and+evaluation+of+the+enzyme-mediated+tryptophan+degradation+in+its+anticellular+activity.">Mechanism of interferon-gamma action. Characterization of indoleamine 2,3-dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity.</a> Journal of Biological Chemistry. 263 (4), 2041-2048 (1988).</li><li>Park, 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=Indoleamine-2,3-dioxygenase+in+thyroid+cancer+cells+suppresses+natural+killer+cell+function+by+inhibiting+NKG2D+and+NKp46+expression+via+STAT+signaling+pathways.">Indoleamine-2,3-dioxygenase in thyroid cancer cells suppresses natural killer cell function by inhibiting NKG2D and NKp46 expression via STAT signaling pathways.</a> Journal of Clinical Medicine. 8 (6), 842-859 (2019).</li><li>Sadok, I., Gamian, A., Staniszewska, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+analysis+of+tryptophan+metabolites.">Chromatographic analysis of tryptophan metabolites.</a> Journal of Separation Science. 40 (15), 3020-3045 (2017).</li><li>Fuertig, R., 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=LC-MS/MS-based+quantification+of+kynurenine+metabolites,+tryptophan,+monoamines+and+neopterin+in+plasma,+cerebrospinal+fluid+and+brain.">LC-MS/MS-based quantification of kynurenine metabolites, tryptophan, monoamines and neopterin in plasma, cerebrospinal fluid and brain.</a> Bioanalysis. 8 (18), 1903-1917 (2016).</li><li>Marcos, J., 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=Targeting+tryptophan+and+tyrosine+metabolism+by+liquid+chromatography+tandem+mass+spectrometry.">Targeting tryptophan and tyrosine metabolism by liquid chromatography tandem mass spectrometry.</a> Journal of Chromatography A. 1434, 91-101 (2016).</li><li>M&#246;ller, M., Du Preez, J., Harvey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+single+analytical+method+for+the+determination+of+tryptophan,+and+its+kynurenine+metabolites+in+rat+plasma.">Development and validation of a single analytical method for the determination of tryptophan, and its kynurenine metabolites in rat plasma.</a> Journal of Chromatography B: Analytical Technologies in Biomedical Life Science. 898, 121-129 (2012).</li><li>Lesniak, W. G., 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=Concurrent+quantification+of+tryptophan+and+its+major+metabolites.">Concurrent quantification of tryptophan and its major metabolites.</a> Analytical Biochemistry. 443 (2), 222-231 (2013).</li><li>Badawy, A. A. B., Morgan, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isocratic+liquid+chromatographic+separation+and+quantification+of+tryptophan+and+six+kynurenine+metabolites+in+biological+samples+with+ultraviolet+and+fluorimetric+detection.">Rapid isocratic liquid chromatographic separation and quantification of tryptophan and six kynurenine metabolites in biological samples with ultraviolet and fluorimetric detection.</a> International Journal of Tryptophan Research. 3, 175-186 (2010).</li><li>Vignau, J., Jacquemont, M. C., Lefort, A., Imbenotte, M., Lhermitte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+tryptophan+and+kynurenine+in+serum+by+HPLC+with+UV+and+fluorescence+detection.">Simultaneous determination of tryptophan and kynurenine in serum by HPLC with UV and fluorescence detection.</a> Biomedical Chromatography. 18 (10), 872-874 (2004).</li><li>Zhang, A., Rijal, K., Ng, S. K., Ravid, K., Chitalia, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mass+spectrometric+method+for+quantification+of+tryptophan-derived+uremic+solutes+in+human+serum.">A mass spectrometric method for quantification of tryptophan-derived uremic solutes in human serum.</a> Journal of Biological Methods. 4 (3), 75 (2017).</li><li>Arnhard, 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=A+validated+liquid+chromatography-high+resolution-tandem+mass+spectrometry+method+for+the+simultaneous+quantitation+of+tryptophan,+kynurenine,+kynurenic+acid,+and+quinolinic+acid+in+human+plasma.">A validated liquid chromatography-high resolution-tandem mass spectrometry method for the simultaneous quantitation of tryptophan, kynurenine, kynurenic acid, and quinolinic acid in human plasma.</a> Electrophoresis. 39 (9-10), 1171-1180 (2018).</li><li>Oh, J. S., Seo, H. S., Kim, K. H., Pyo, H., Chung, B. C., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+profiling+of+tryptophan+and+its+related+metabolites+in+patients+with+metabolic+syndrome+by+liquid+chromatography-electrospray+ionization/mass+spectrometry.">Urinary profiling of tryptophan and its related metabolites in patients with metabolic syndrome by liquid chromatography-electrospray ionization/mass spectrometry.</a> Analytical and Bioanalytical Chemistry. 409 (23), 5501-5512 (2017).</li><li>Huang, 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=A+simple+LC-MS/MS+method+for+determination+of+kynurenine+and+tryptophan+concentrations+in+human+plasma+from+HIV-infected+patients.">A simple LC-MS/MS method for determination of kynurenine and tryptophan concentrations in human plasma from HIV-infected patients.</a> Bioanalysis. 5 (11), 1397-1407 (2013).</li><li>Yamada, K., Miyazzaki, T., Shibata, T., Hara, N., Tsuchiya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+tryptophan+and+related+compounds+by+liquid+chromatography/electrospray+ionization+tandem+mass+spectrometry.">Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry.</a> Journal of Chromatography B: Analytical Technologies in Biomedical and Life Sciences. 867 (1), 57-61 (2008).</li><li>Zhu, W., 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=Quantitative+profiling+of+tryptophan+metabolites+in+serum,+urine,+and+cell+culture+supernatants+by+liquid+chromatography-tandem+mass+spectrometry.">Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry.</a> Analytical and Bioanalytical Chemistry. 401, 3249-3261 (2011).</li><li>Hashioka, S., 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=Metabolomics+analysis+implies+noninvolvement+of+the+kynurenine+pathway+neurotoxins+in+the+interferon-gamma-+induced+neurotoxicity+of+adult+human+astrocytes.">Metabolomics analysis implies noninvolvement of the kynurenine pathway neurotoxins in the interferon-gamma- induced neurotoxicity of adult human astrocytes.</a> Neuropsychiatry. 7 (2), (2017).</li><li>Sadok, I., Rachwa&#322;, K., Staniszewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+optimized+and+validated+LC-MS+method+for+simultaneous+quantification+of+tryptophan+metabolites+in+culture+medium+from+cancer+cells.">Application of the optimized and validated LC-MS method for simultaneous quantification of tryptophan metabolites in culture medium from cancer cells.</a> Journal of Pharmaceutical and Biomedical Analysis. 176, 112805-112815 (2019).</li><li>Annesley, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+suppression+in+mass+spectrometry.">Ion suppression in mass spectrometry.</a> Clinical Chemistry. 49 (7), 1041-1044 (2003).</li><li>Hou&#233;e-L&#233;vin, C., 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=Exploring+oxidative+modifications+of+tyrosine:+An+update+on+mechanisms+of+formation,+advances+in+analysis+and+biological+consequences.">Exploring oxidative modifications of tyrosine: An update on mechanisms of formation, advances in analysis and biological consequences.</a> Free Radical Research. 49 (4), 347-373 (2015).</li><li>Wei, H., 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=Abnormal+Expression+of+Indoleamine+2,+3-Dioxygenase+in+Human+Recurrent+Miscarriage.">Abnormal Expression of Indoleamine 2, 3-Dioxygenase in Human Recurrent Miscarriage.</a> Reproductive Sciences. , (2019).</li><li>Liu, 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=Expression+of+indoleamine+2,3-dioxygenase+in+nasopharyngeal+carcinoma+impairs+the+cytolytic+function+of+peripheral+blood+lymphocytes.">Expression of indoleamine 2,3-dioxygenase in nasopharyngeal carcinoma impairs the cytolytic function of peripheral blood lymphocytes.</a> BMC Cancer. 9, 416 (2009).</li><li>Mailankot, 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=Indoleamine+2,3-dioxygenase+overexpression+causes+kynurenine-modification+of+proteins,+fiber+cell+apoptosis+and+cataract+formation+in+the+mouse+lens.">Indoleamine 2,3-dioxygenase overexpression causes kynurenine-modification of proteins, fiber cell apoptosis and cataract formation in the mouse lens.</a> Laboratory Investigation: A Journal of Technical Methods and Pathology. 89 (5), 498-512 (2009).</li></ol>

著者らは開示するものは何もない。

本論文では、 インビトロ 培養ヒト癌細胞から培地で測定される4つの主要Trp代謝物(3HKyn、Kyn、3HAA、XA)の同時定量化のための詳細なLC-SQプロトコルを提示する。分析の中で最も重要な点であるサンプル調製、クロマトグラフィー/質量分析法、データ解釈に特に注意が払われます。
一般に、LC-MS分析は、高感度のため、使用材料のプロトコルの厳密さと純度の最高水?...

キヌレニン経路(KP)は、ヒト細胞におけるトリプトファン(Trp)の変性の主要な経路である。インドールアミン-2,3-ジオキシゲナーゼ(IDO-1)は、KPの第1及び制限酵素であり、TrpをN-フォルミルカヌレニン1に変換する。KP内のさらなるステップは、他の二次代謝産物、すなわち種々の生物学的特性を示すキヌレニンを生成する。キヌレニン(Kyn)は、アリル炭化水素受容体(AhR)2に結合した後に、毒性特性および細胞イベントを調節する第1の安定したTrpカタボライトである。その後、Kynは自発的にまたは酵素媒介プロセス内のいくつかの分子に変換され、3-ヒドロキシキヌレニン(3HKyn)、アントラニル酸(AA)、3-ヒドロキシアントラニル酸(3-HAA)、キヌレン酸(Kyna)、キナンテレン酸(XA)などの代謝産物を生成する。別の下流代謝産物は、2-アミノ-3-カルボキシムコン酸-6-セミアルデヒド(ACMS)、キノリン酸(QA)またはピコリン酸(PA)1に非酵素的なサイクライゼーションを行う。最後に、QAは、さらにニコチンアミドアデニンジヌクレオチド(NAD+)3、重要な酵素補因子であるKP終点代謝産物に変換される。キヌレニンには、キナやPAなどの神経保護特性があるものもあれば、3HAAや3HKynの他の人は有毒である4.3HKynから形成されるキサンスレン酸は抗酸化作用と血管弛緩性5を提示する。XAは、老化したレンズに蓄積し、上皮細胞のアポトーシスを導く6.KPは、20世紀半ばに記述され、様々な障害への関与が実証されたときに、より注目を集めました。この代謝経路の活性の増加およびいくつかのキヌレニンの蓄積は、免疫応答を調節し、うつ病、統合失調症、脳症、HIV、認知症、筋萎縮性側索硬化症、マラリア、アルツハイマー病、ハンチントン病、癌4、7などの異なる病理学的状態に関連している。Trp代謝のいくつかの変化は、腫瘍微小環境および癌細胞2,8で観察される。また、キヌレニンは有望な疾患マーカー9と考えられている。がん研究において、in vitro細胞培養モデルは、抗癌剤10に対する応答の前臨床評価のために十分に確立され、広く使用されている。Trp代謝産物は、細胞によって培養培地中に分泌され、キヌレニン経路の状態を評価するために測定することができる。そのため、容易で柔軟で信頼性の高いプロトコルを備えた様々な生物学的標本において、できるだけ多くのKP代謝産物を同時に検出するための適切な方法を開発する必要があります。
本論文では、がん細胞から採取した培養後の培地において、キヌレニン経路代謝産物であるKyn,3HKyn,3HAA,XAをLC-SQで同時に決定するためのプロトコルについて述べた。現代の分析的アプローチにおいて、液体クロマトグラフィー13、14、15、16は、個々のトリプトファン・カマボリトの同時検出および定量化に好ましく、エーリッヒ試薬11,12を利用した生化学的非特異的アッセイとは対照的である。現在のところ、ヒト検体におけるキヌレニンの測定には、主に紫外線または蛍光検出器13、17、18、19による液体クロマトグラフィーに基づく多くの方法がある。液体クロマトグラフィーと質量分析検出器(LC-MS)を組み合わせると、感度が高く(検出の下限)、選択性、再現性が高いため、このタイプの分析に適しているようです。
Trp代謝産物は、ヒト血清、血漿および尿13、20、21、22、23において既に決定されているが、細胞培養培地のような他の生物学的標本に対する方法も望まれている。以前、LC-MSは、ヒトグリオーマ細胞の培養後に採取した培地中のTrp由来化合物、単球、樹状細胞、またはインターフェロンガンマ(IFN-γ)24、25、26で処理されたアストロサイトに使用した。現在、がんモデルで使用される異なる培養培地、細胞、および治療における複数の代謝産物の評価を可能にする新しい検証済みプロトコルが必要です。
開発された方法の目的は、KPの異常を示すことができる4つの主要なキヌレニンを(1つの分析実行内で)定量することです。ここに提示される、 インビトロ 培養ヒト癌細胞27から採取した培地において、1つの内部標準(3-ニトロチロシン、3NT)を用いて選択された有意義なキヌレニンの定量的LC-SQ分析のための我々の最近発表されたプロトコルの重要なステップが示される。我々の知る限りでは、 インビトロ で増殖した細胞から得られた培養培地中で3HKyn、3HAA、KynおよびXAを同時に定量する初のLC-SQプロトコルです。いくつかの変更では、この方法は、より広い範囲の細胞培養モデルにおけるTrp代謝の変化を研究するためにさらに適用される可能性がある。

<table border="0" cellpadding="0" cellspacing="0" style="width:1360px;" width="1359">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-hydroksy-DL-kynurenina</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">H1771</td>
			<td style="width:513px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-hydroxyanthranilic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">148776</td>
			<td>97%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-nitro-L-tyrosine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">N7389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Acetonitrile</td>
			<td style="width:175px;">Supelco</td>
			<td>1.00029</td>
			<td>hypergrade for LC-MS LiChrosolv</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Activated charcoal</td>
			<td style="width:175px;">Supelco</td>
			<td>05105</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Analytical balance</td>
			<td style="width:175px;">Ohaus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:411px;">Analytical column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>959764-902</td>
			<td>Zorbax Eclipse Plus C18 rapid resolution HT (2.1 x 100 mm, 1.8 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium formate</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">7022</td>
			<td>eluent additive for LC-MS, LiChropur, &#8805;99.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td style="width:175px;">Sigma</td>
			<td>1001887398</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caps for chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5185-5820</td>
			<td>blue screw caps,PTFE/red sil sepa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dish</td>
			<td style="width:175px;">Nest</td>
			<td>704004</td>
			<td>polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture plate</td>
			<td style="width:175px;">Biologix</td>
			<td>07-6012</td>
			<td>12-well, non-pyrogenic, non-cytoxic, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>HERAcell 150i Cu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5415R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-2278</td>
			<td>Eppendorf type, 1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-3693</td>
			<td>Falcon type, 50 mL, PP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic data acqusition and analysis software</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td></td>
			<td>LC/MSD ChemStation (B.04.03-S92)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic insert vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-1387</td>
			<td>100 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5182-0714</td>
			<td>2 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dimethyl sulfoxide</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">1.02950</td>
			<td>Uvasol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dubelcco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:175px;">PAN Biotech</td>
			<td>P04-41450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dual meter pH/conductivity</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td style="width:261px;"></td>
			<td>SevenMulti</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evaporator</td>
			<td style="width:175px;">Genevac</td>
			<td style="width:261px;"></td>
			<td>model EZ-2.3 Elite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Formic acid</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">5.33002</td>
			<td>for LC-MS LiChropur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glass bottle for reagents storage</td>
			<td style="width:175px;">Bionovo</td>
			<td>S-2070</td>
			<td>50 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Guard column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">959757-902</td>
			<td>Zorbax Eclipse Plus-C18 Narrow Bore Guard Column (2.1 x 12.5 mm, 5 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hydrochloric acid</td>
			<td style="width:175px;">Merck</td>
			<td>1.00317.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">L-kynurenine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">K8625</td>
			<td>&#8805;98% (HPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Magnetic stirrer</td>
			<td style="width:175px;">Wigo</td>
			<td style="width:261px;"></td>
			<td>ES 21 H</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microbalance</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td></td>
			<td>model XP6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Milli-Q system</td>
			<td style="width:175px;">Millipore</td>
			<td>ZRQSVPO30</td>
			<td>Direct-Q 3 UV with Pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quadrupole mass spectrometer</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>G1948B</td>
			<td>model 6120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:175px;">Sigma-Adrich</td>
			<td>048M4874V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td style="width:175px;">Bio Tek</td>
			<td></td>
			<td>Synergy 2 operated by Gen5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td style="width:175px;">Merck</td>
			<td>1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium dihydrogen phosphate</td>
			<td style="width:175px;">Merck</td>
			<td>1.05108.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Assay Dye Reagent Concentrate</td>
			<td style="width:175px;">BioRad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">See-saw rocker</td>
			<td style="width:175px;">Stuart</td>
			<td>SSL4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipette</td>
			<td style="width:175px;">Nest</td>
			<td>326001</td>
			<td>5 mL, polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td style="width:175px;">Merck</td>
			<td>1.06346.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent inlet filter</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5041-2168</td>
			<td>glass filter, 20 &#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6524</td>
			<td>1 L, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6526</td>
			<td>1 L, amber glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spreadsheet program</td>
			<td style="width:175px;">Microsoft Office</td>
			<td></td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stir bar</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-2003</td>
			<td>teflon coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for culture medium filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>7-8803</td>
			<td>regenerated cellulose, &#216; 30 mm, 0,45 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for mobile phase components filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-0018</td>
			<td>nylon, &#216; 30 mm, 0,22 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td style="width:175px;">VWR</td>
			<td>10062-900</td>
			<td>96-wells, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA Solution</td>
			<td style="width:175px;">Sigma-Aldrih</td>
			<td>T4049-100 mL</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:411px;">Ultra-High Performance Liquid Chromatography system</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">G1367D, G1379B, G1312B, G1316C</td>
			<td style="width:513px;">1200 Infinity system consisted of autosampler (G1367D), degasser (G1379B), binary pump (G1312B), column thermostat (G1316C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasound bath</td>
			<td>Polsonic</td>
			<td>104533</td>
			<td>model 6D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>Biosan</td>
			<td></td>
			<td>model V-1 plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xanthurenic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">D120804</td>
			<td>96%</td>
		</tr>
	</tbody>
</table>

simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures

キヌレニン経路とキヌレニンと呼ばれるトリプトファンのカマボリテは、免疫調節と癌生物学への関与に対する注目度が高まっています。 インビトロ 細胞培養アッセイは、疾患メカニズムにおける異なるトリプトファンカマボリテの寄与について学び、治療戦略をテストするためにしばしば使用される。分泌代謝物およびシグナル伝達分子が豊富な細胞培養培地は、トリプトファン代謝および他の細胞事象の状況を反映する。複雑な細胞培養培地における複数のキヌレニンの信頼性の高い定量のための新しいプロトコルは、複数のサンプルの信頼性と迅速な分析を可能にするために望まれる。これは、質量分析と結合された液体クロマトグラフィーで達成することができる。この強力な技術は、代謝産物の定量のために多くの臨床および研究機関で使用され、キヌレニンの測定に使用することができます。
ここでは、4つのキヌレニン、すなわちキヌレニン、3-ヒドロキシキヌレニン、3-ヒドロキシキヌレニン、3-ヒドロキシアントラニル、およびキサンスレン酸を 、インビトロ 培養癌細胞から採取した培地において、単四重極質量分析(LC-SQ)と結合した液体クロマトグラフィーの使用を示す。SQ検出器は、他の質量分析計に比べて使いやすく、安価です。SQ-MS分析では、サンプルから複数のイオンが生成され、特定の質量電荷比(m/z)に従って分離され、その後に単一イオン監視(SIM)モードを使用して検出されます。
本稿では、報告された方法の利点を取り上げ、いくつかの弱点を示す。これは、クロマトグラフィーおよび質量分析と共にサンプル調製を含むLC-SQ分析の重要な要素に焦点を当てています。品質管理、方法キャリブレーション条件、マトリックス効果の問題についても説明します。我々は、すべての標的検体のための1つのアナログ標準として3-ニトロチロシンの簡単な適用を記述した。ヒト卵巣および乳癌細胞の実験によって確認されるように、提案されたLC-SQ法は信頼できる結果を生成し、さらに他 のインビトロ 細胞モデルに適用することができる。

LC-MSは、複雑な標本の一部の成分がいわゆるマトリックス効果を引き起こし、分析物のイオン化を損なうにもかかわらず、生物学的に活性な分子の定量化において紛れもない利点を提示する。それはイオン抑制またはイオン増強をもたらし、精度を強く低下させ、LC-MSの検出/定量の限界に影響を及ぼし、これは方法の「弱い」点と考えられる。我々のプロトコルでは、イオンは正のモードで?...

Watch this Scientific Journal Video about Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures at JoVE.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

ここで説明するキヌレニン経路で生成される4種類のトリプトファン代謝産物(キヌレニン、3−ヒドロキシキヌレニン、キサンチュレン酸、3−ヒドロキシアントラニル酸)を、液体クロマトグラフィーによって分析した培養液から採取した培地中の単一の四重極質量分析法を用いて説明する。

癌細胞培養から採取した培地における液体クロマトグラフィー-質量分析法による選択キヌレニンの同時定量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

simultaneous-quantification-selected-kynurenines-analyzed-liquid

Research

JoVE Journal

Bioengineering

3.3K ビュー.  The John Paul II Catholic University of Lublin. ここで説明するキヌレニン経路で生成される4種類のトリプトファン代謝産物(キヌレニン、3−ヒドロキシキヌレニン、キサンチュレン酸、3−ヒドロキシアントラニル酸)を、液体クロマトグラフィーによって分析した培養液から採取した培地中の単一の四重極質量分析法を用いて説明する。

ビデオ: Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

生物工学

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

パラメータ化拡張期充満フォーマリズムを経由Transmitral流れの運動学的モデルベースの​​解析によるグローバル拡張期機能の定量化

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

3次元細胞培養モデルの質量分析イメージング用試料調製の戦略

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

生物学的液体培地中でスケーラブルな、メタリック高アスペクト比のナノコンポジットの生成

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

マイクロ コント ローラーは従来のタバコの煙、電子たばこエアロゾル液体抽出物の世代のデバイスを運営

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

ナノ製剤中のエンドトキシン検出カブトガニ リムラス ライセート (ラル) 試金を使用して

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

マイクロ流体チップを使用した決定論的方法で単一細胞ベースの共培養を確立する

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Methods Article

癌細胞培養から採取した培地における液体クロマトグラフィー-質量分析法による選択キヌレニンの同時定量化