Name: simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures
Uploaded: 2020-05-09T13:30:00
Description: 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.

Dette arbeidet ble støttet av EU fra European Regional Development Fund under Operasjonell programutvikling i Øst-Polen 2007-2013 (POPW.01.03.00-06-003/09-00), av Fakultet for bioteknologi og miljøvitenskap ved John Paul II Catholic University of Lublin (Young Scientists grant for Ilona Sadok), Polish National Science Centre, OPUS13 (2017/25/B/NZ4/01198 for Magdalena Staniszewska, prinsippetterforsker).  Forfatterne takker prof. Agnieszka Ścibior fra Laboratory of Oxidative Stress, Centre for Interdisciplinary Research, JPII CUL for å dele utstyret for celle culturing og protein kvantifisering.

1. Utarbeidelse av standard 3NT, Kyn, 3HKyn, 3HAA, XA standard lagerløsninger
<ol><li>Vei reagensene i et hetteglass med høyest nøyaktighet (0,3 mg hver). For bedre nøyaktighet, skaler opp reagensene, justere volumet av oppløsningsvæsken i henhold til trinn 1.2.</li>
	<li>Oppløs reagensene i 300 μL av oppløsningsvæsken for å oppnå en lagerløsning på 1 g/l. Løs opp 3NT i 1 % (v/v) maursyre (FA) i vann; Kyn, 3HAA og XA i dimetylsulfoksid (DMSO); 3HKyn i vann surgjort til pH 2.5 med saltsyre (HCl). FORSI...

<ol>
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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. 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Dette papiret presenterer en detaljert LC-SQ-protokoll for samtidig kvantifisering av fire store Trp-metabolitter (3HKyn, Kyn, 3HAA, XA) som måles i et medium fra in vitro kultiverte humane kreftceller. Spesiell oppmerksomhet er betalt til prøveforberedelsen, kromatografisk / massespektrometriprosedyre og datatolkning, de viktigste punktene i analysen.
Generelt krever LC-MS-analyse, på grunn av høy følsomhet, de høyeste standardene for en protokollseshet og renhet av brukte mate...

Kynurenine pathway (KP) er den viktigste ruten for tryptofan (Trp) katabolisme i menneskelige celler. Indoleamin-2,3-dioksygenase (IDO-1) i ekstrahepatiske celler er det første og begrensende enzymet av KP og konverterer Trp til N-formylkynurenine1. Ytterligere trinn i KP genererer andre sekundære metabolitter, nemlig kynurenines som viser ulike biologiske egenskaper. Kynurenine (Kyn) er den første stabile Trp catabolite som viser giftige egenskaper og regulerer cellulære hendelser etter binding til arylhydrokarbonreseptoren (AhR)2. Deretter blir Kyn forvandlet til flere molekyler enten spontant eller i enzymmedierte prosesser, og genererer slike metabolitter som 3-hydroksykynurenin (3HKyn), antranilic acid (AA), 3-hydroksyantranisyre (3-HAA), kynurensyre (Kyna) og xanthurenic acid (XA). En annen nedstrøms metabolitt, 2-amino-3-karboksymukonsyre-6-semialdehyd (ACMS), gjennomgår ikke-enzymatisk syklonisering til quinolinic acid (QA) eller picolinsyre (PA)1. Til slutt blir QA videre forvandlet til nikotinamid-adenin dinucleotide (NAD+)3,KP sluttpunktmetabolitten som er en viktig enzymkofaktor. Noen kynureniner har nevrobeskyttende egenskaper som Kyna og PA, mens de andre, det vil at 3HAA og 3HKyn er giftige4. Xanthurenic acid, som er dannet av 3HKyn, presenterer antioksidant og vasorelaxation egenskaper5. XA akkumuleres i aldrende linser og fører til apoptose av epitelceller6. KP, beskrevet i midten av 1900-tallet, fikk mer oppmerksomhet da dens engasjement i ulike lidelser ble demonstrert. Økt aktivitet av denne metabolske ruten og akkumulering av noen kynureniner modulerer immunresponsen og er forbundet med forskjellige patologiske tilstander som depresjon, schizofreni, encefalopati, HIV, demens, amyotrofisk lateral sklerose, malaria, Alzheimers, Huntingtons sykdom og kreft4,7. Noen endringer i Trp metabolisme observeres i tumor mikromiljøer og kreftceller2,8. Videre anses kynurenines som lovende sykdomsmarkører9. I kreftforskning er in vitro cellekulturmodeller godt etablert og mye brukt til preklinisk evaluering av respons på kreftmedisiner10. Trp metabolitter utskilles av celler i kulturmediet og kan måles for å vurdere statusen til kynureninebanen. Derfor er det behov for å utvikle egnede metoder for samtidig påvisning av så mange KP-metabolitter som mulig i en rekke biologiske prøver med en enkel, fleksibel og pålitelig protokoll.
I denne artikkelen beskriver vi en protokoll for samtidig bestemmelse av fire kynureninveimetabolitter: Kyn, 3HKyn, 3HAA og XA av LC-SQ i et postkulturmedium samlet inn fra kreftceller. I en moderne analytisk tilnærming foretrekkes flytende kromatografi13,14,15,16 for den samtidige deteksjon og kvantifisering av de enkelte tryptofan katabolitter, i motsetning til biokjemiske uspesifikke analyser som bruker Ehrlich reagens11,12. I dag er det mange metoder tilgjengelig for kynurenines bestemmelse i menneskelige prøver, hovedsakelig basert på flytende kromatografi med ultrafiolett eller fluorescensdetektorer 13,17,18,19. Flytende kromatografi kombinert med en massespektrometridetektor (LC-MS) virker mer egnet for denne typen analyse, på grunn av deres høyere følsomhet (nedre grenser for deteksjon), selektivitet og repeterbarhet.
Trp metabolitter har allerede blitt bestemt i humant serum, plasma og urin13,20,21,22,23,men metodene for andre biologiske prøver, som cellekultur medium er også ønsket. Tidligere ble LC-MS brukt til Trp-avledede forbindelser i et medium samlet etter dyrking av humane gliomceller, monocytter, dendrittiske celler eller astrocytter behandlet med interferon gamma (IFN-γ)24,25,26. For tiden er det behov for nye validerte protokoller som kan tillate en vurdering av flere metabolitter i ulike kulturmedier, celler og behandlinger som brukes i kreftmodeller.
Formålet med den utviklede metoden er å kvantifisere (innen ett analytisk løp) fire store kynureniner som kan indikere abnormiteter i KP. Presentert her er kritiske trinn i vår nylig publiserte protokoll for kvantitativ LC-SQ analyse av utvalgte meningsfulle kynureniner ved hjelp av en intern standard (3-nitrotyrosin, 3NT) i mediet samlet inn fra in vitro kultiverte humane kreftceller27. Så vidt vi vet, er det den første LC-SQ-protokollen for samtidig kvantifisering av 3HKyn, 3HAA, Kyn og XA i et kulturmedium hentet fra de in vitro dyrkede cellene. Ved noen modifikasjoner, metoden kan brukes videre for å studere endringene i Trp metabolisme i et bredere spekter av celle kultur modeller.

<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

Kynurenine banen og tryptofan katabolitter kalt kynurenines har fått økt oppmerksomhet for deres engasjement i immunregulering og kreftbiologi. En in vitro celle kultur analyse brukes ofte til å lære om bidraget fra ulike tryptofan katabolitter i en sykdomsmekanisme og for å teste terapeutiske strategier. Cellekulturmedium som er rik på utskillede metabolitter og signalmolekyler gjenspeiler statusen til tryptofanmetabolismen og andre cellulære hendelser. Nye protokoller for pålitelig kvantifisering av flere kynureniner i det komplekse cellekulturmediet er ønsket å tillate en pålitelig og rask analyse av flere prøver. Dette kan oppnås med flytende kromatografi kombinert med massespektrometri. Denne kraftige teknikken er ansatt i mange kliniske og forskningslaboratorier for kvantifisering av metabolitter og kan brukes til å måle kynurenines.
Presentert her er bruk av flytende kromatografi kombinert med enkelt quadrupole massespektrometri (LC-SQ) for samtidig bestemmelse av fire kynurenines, det vil si kynurenine, 3-hydroxykynurenine, 3-hydroksyantranilic, og xanthurenic syre i mediet samlet fra in vitro kultiverte kreftceller. SQ detektor er enkel å bruke og billigere i forhold til andre massespektrometre. I SQ-MS-analysen genereres flere ioner fra prøven og skilles i henhold til deres spesifikke masse-til-lade-forhold (m/z), etterfulgt av gjenkjenningen ved hjelp av en SIM-modus (Single Ion Monitoring).
Dette papiret trekker oppmerksomheten på fordelene med den rapporterte metoden og indikerer noen svake punkter. Det er fokusert på kritiske elementer av LC-SQ analyse inkludert prøve forberedelse sammen med kromatografi og massespektrometri analyse. Kvalitetskontroll, metodekalibreringsforhold og matriseeffektproblemer diskuteres også. Vi beskrev en enkel anvendelse av 3-nitrotyrosin som en analog standard for alle målanalytter. Som bekreftet av eksperimenter med humane eggstokk- og brystkreftceller, genererer den foreslåtte LC-SQ-metoden pålitelige resultater og kan videre brukes på andre in vitro cellulære modeller.

LC-MS gir udiskutable fordeler i kvantifiseringen av biologisk aktive molekyler, selv om noen komponenter av komplekse prøver forårsaker såkalte matriseeffekter og kompromissionisering av analytter. Det fører til ionundertrykkelse eller ionforbedring, sterkt avtagende nøyaktighet og påvirker en grense for deteksjon / kvantifisering av LC-MS, som regnes som et "'svakt" punkt av metoden. I vår protokoll genereres ionene ved elektrosprayionisering (ESI) i positiv modus, som også ble brukt i andre studier på Trp-met...

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

Beskrevet her er en protokoll for bestemmelse av fire forskjellige tryptofan metabolitter generert i kynurenin banen (kynurenine, 3-hydroxykynurenine, xanthurenic syre, 3-hydroksyantranisyre) i mediet samlet inn fra kreftcellekulturer analysert av flytende kromatografi kombinert med en enkelt quadrupole massespektrometri.

Samtidig kvantifisering av utvalgte kynureniner analysert av flytende kromatografi-massespektrometri i medium samlet fra kreftcellekulturer

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

Research

JoVE Journal

Bioengineering

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

Cellular Lipid Uttak for Målrettet Stabil isotopfortynning 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 ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Avbildning av biologisk vev ved Desorpsjon Elektrospray Ionization massespektrometri

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Shock Wave Application to Cell Cultures

Shock Wave Søknad til cellekulturer

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

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

Kvantifisering av Global diastolisk funksjon av Kinetisk modellering basert analyse av transmitral Flow via Parametrized diastolisk Fylling formalisme

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

Prøvepreparering Strategier for massespektrometri Imaging av 3D-Cell Kultur Modeller

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

Generering av skalerbare, Metallic høy Aspect Ratio Nanocomposites i en biologisk væske 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 ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Automatisert robot flytende håndtering samlingen av modulære DNA enheter

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

Syntese av Biokompatible Liquid Crystal Elastomer Skum som Cell Stillas for 3D Spatial cellekulturer

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 ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

Produksjon av genmodifiserte Golden syrer Hamsters ved Pronuclear injeksjon av CRISPR/Cas9 komplekset

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

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

En Microcontroller drives enhet for generering av flytende ekstrakter fra konvensjonelle sigarett røyk og elektronisk sigarett Aerosol

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