This work was supported by the European Union from European Regional Development Fund under the Operational Programme Development of Eastern Poland 2007-2013 (POPW.01.03.00-06-003/09-00), by Faculty of Biotechnology and Environmental Sciences of the 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, Principle Investigator).&#160; The authors thank Prof. Agnieszka &#346;cibior from the Laboratory of Oxidative Stress, Centre for Interdisciplinary Research, JPII CUL for sharing the equipment for cell culturing and protein quantification.&#160;

1. Preparation of standard 3NT, Kyn, 3HKyn, 3HAA, XA standard stock solutions
<ol>
	<li>Weigh the reagents in a vial with the highest accuracy (0.3 mg each). For better accuracy, scale up the reagents, adjusting the volume of the solvent according to step 1.2.</li>
	<li>Dissolve the reagents in 300 &#181;L of the solvent to obtain a stock solution of 1 g/L. Dissolve 3NT in 1% (v/v) formic acid (FA) in water; Kyn, 3HAA, and XA in dimethyl sulfoxide (DMSO); 3HKyn in water acidified to pH 2.5 with hydrochloric acid (HCl). 
	CAUTION: 3HAA irritates eyes, nose, throat and lungs. It is harmful when inhaled, in contact with skin, and if swallowed. Wear appropriate protection i.e., gloves and mask.</li>
	<li>Tightly close the vial and place it in an ultrasonic bath for 1 min to accelerate dissolution. 
	NOTE: Store the stock solutions at -20 &#176;C and minimize freezing/thawing (3 cycles max) due to the instability of the stock solutions.</li>
</ol>
2. Preparation of charcoal treated culture medium
<ol>
	<li>In a tube, weigh 280 mg of activated charcoal and add 5 mL of the liquid medium prepared for culturing the cells of interest.</li>
	<li>Shake the tube with the medium and charcoal on a see saw rocker for 2 h, at room temperature (set speed to 50 oscillations/min). Next, centrifuge&#160;the tubes at 6000 x g for 15 min.</li>
	<li>Remove the tube from the centrifuge and carefully collect the supernatant without disturbing the sediment. Repeat centrifugation if necessary, to remove all charcoal residues.</li>
	<li>Filter the supernatant using a 0.45 &#181;m syringe filter.</li>
	<li>Ensure that the charcoal pretreated culture medium is deprived of kynurenines traces by running a pilot sample on LC-MS as described in step 6.3.2. Otherwise, repeat steps 2.1-2.4. 
	NOTE: If the complete culture medium initially does not contain kynurenines, purification step using charcoal might be omitted. In this case, prepare calibration standards and quality control samples using the complete medium usually prepared for cell culturing.</li>
	<li>Store the prepared medium at 4 &#176;C until analysis.</li>
</ol>
3. Making the calibration solutions and calibration curves
<ol>
	<li>Spike the charcoal pretreated culture medium with 0.75 &#181;L of 0.1 g/L 3NT solution and add four standards (3HKyn, 3HAA, Kyn, XA) at least at six different concentrations to cover calibration ranges. Keep the final volume of each sample at 150 &#181;L. Vortex well. Use 1.5 mL centrifuge tubes for sample preparation. 
	NOTE: Suggested calibration points for 3HKyn: 0.018, 0.045, 0.22, 1.12, 2.23, 4.46 &#181;mol/L; for Kyn: 0.0096, 0.048, 0.24, 1.20, 2.40, 3.84 &#181;mol/L; for 3HAA: 0.033, 0.16, 0.65, 3.27, 6.53, 13.06 &#181;mol/L; for XA: 0.019, 0.13, 0.49, 1.22, 3.65, 4.87 &#181;mol/L.</li>
	<li>Add 150 &#181;L of cold methanol (kept at -20 &#176;C) containing 1% (v/v) formic acid into each tube for sample deproteinization. Tightly close the tubes and vortex well.</li>
	<li>Incubate the samples at -20 &#176;C for 40 min.</li>
	<li>Centrifuge the samples at 14,000 x g for 15 min at 4 &#176;C. Remove the tubes from the centrifuge. Collect supernatants into the new tubes. Do not disturb the sediment.</li>
	<li>Transfer 270 &#181;L of the supernatant into the glass vial using an automatic pipette. Put the vials into the evaporator and gently evaporate until dry. Use the appropriate program for water/methanol fractions to evaporate volatile components. Do not apply temperature higher than 40 &#176;C and&#160;avoid over-drying. 
	NOTE: Flat bottom glass vials, i.e., chromatographic vials facilitate faster evaporation and higher recoveries in comparison to plastic conical tubes.</li>
	<li>After 30 min, check the evaporation status. If necessary, continue evaporation (e.g., add extra 10 min). Avoid over-drying.</li>
	<li>Remove the vials from the evaporator. Reconstitute each sample in 60 &#181;L of 0.1% (v/v) formic acid in water. Add the solvent into the vial containing the residual material. Tightly close the vials and vortex-well.</li>
	<li>Transfer the sample into a 1.5 mL tube and spin at 14,000 x g for 15 min at 4 &#176;C to separate the precipitated protein.</li>
	<li>Without disturbing the protein pellet, transfer the supernatants into chromatographic vials with conical glass inserts with an automatic pipette. Tightly close the vials.</li>
	<li>Check for the presence of air bubbles in the insert vial and remove them if necessary (e.g., by vortexing).</li>
	<li>Transfer the chromatographic vials containing samples into LC autosampler. Record the position of samples placed in an autosampler tray.</li>
</ol>
4. Preparation of quality control (QC) samples
<ol>
	<li>Spike the charcoal-pretreated culture medium (prepared in a 1.5 ml centrifugal tube) with 0.75 &#181;L of 0.1 g/L 3NT solution and standards of four kynurenines (3HKyn, 3HAA, Kyn, XA) at one concentration selected from the linear range of the calibration curves. Keep the final volume of the sample at 150 &#181;L. 
	NOTE: If applicable, prepare several QC samples at different concentrations falling under the linear range of the calibration curve.</li>
	<li>Follow the protocol described in section 3 (steps 3.2-3.11).</li>
</ol>
5. Setting up the LC-MS system
<ol>
	<li>Prepare the mobile phase solvents: Solvent A: 20 mmol/L ammonium formate in ultrapure water (pH 4.3 adjusted with formic acid); Solvent B: 100% acetonitrile. 
	NOTE: Use borosilicate glass bottles only. Rinse bottles with ultrapure water before refilling it. Do not use bottles cleaned with detergents. 
	CAUTION: Acetonitrile causes severe health effects or death. It is easily ignited by heat. Acetonitrile liquid and vapor can irritate eyes, nose, throat and lungs.
	<ol>
		<li>Prepare 5 mol/L stock solution of ammonium formate by dissolving a crystalline reagent (15.77 g) in 50 mL of ultrapure water in a glass bottle. Stir until all residuals dissolve. Filter through the 0.45 &#181;m membrane to remove any residual debris (e.g., by using a nylon syringe filter). Store the stock solution at 4 &#176;C.</li>
		<li>Prepare Solvent A by adding 4 mL of 5 mol/L ammonium formate in water to 980 mL of ultrapure water in an amber glass bottle and stir well. Immerse a stirring bar and put the bottle with the solvent on a magnetic stirrer. Immerse a pH electrode into the solution and control the pH under stirring. Add formic acid stock solution (98%-100%) dropwise using an automatic pipette with 0.2 mL tip to obtain pH 4.3, adjust the volume up to 1 L with ultrapure water and stir well. 
		NOTE: Prepare the solvents at least once a week to prevent any microbial growth. 
		CAUTION: Formic acid (FA) is toxic when inhaled, causes skin burns and eye damage. Work under the fume hood; wear protective gloves and coat.</li>
		<li>Filter all solutions through the 0.22 &#181;m membrane (e.g., nylon syringe filter) to remove any residual debris. Optionally, use the solvent inlet filters dedicated for LC solvent reservoirs to protect the system from incoming particles.</li>
	</ol>
	</li>
	<li>Start LC-MS control and data acquisition software.</li>
	<li>Purge the LC system with the mobile phase to remove bubbles, and to prime all solvent channels.</li>
	<li>Ensemble a guard column to protect the analytical column from clogging.</li>
	<li>Flush the guard and analytical column (C18, 2.1 x 100 mm, 1.8 &#181;m) with 100% acetonitrile for about 30 min, and then with 100% solvent A until a stable pressure in the column is observed. Control the system performance using the control software.</li>
	<li>Set the appropriate LC parameters in the data acquisition software.
	<ol>
		<li>Set up the gradient program: 0-8 min solvent B 0%; 8-17 min solvent B 0-2%; 17-20 min solvent B 2%; 20-32 min solvent B 10-30%; 32-45 min solvent B 0%.</li>
		<li>Set the mobile phase flow rate at 0.15 mL/min, total analysis run time for 45 min, column temperature at +40 &#176;C and injection volume at 10 &#181;L.</li>
	</ol>
	</li>
	<li>Set the acquisition parameters of the mass spectrometry detector.
	<ol>
		<li>Apply ionization parameters: single ion monitoring (SIM), positive ionization, nebulizer pressure of 50 psi, +350 &#176;C drying gas temperature, drying gas flow of 12 L/min, and 5500 V capillary voltage.</li>
		<li>Select the analyte monitoring ions: for 3HKyn m/z 225.0 (scan period: 2-6 min, fragmentor voltage: 100 V); for Kyn m/z 209.0 (scan period: 6-12 min, fragmentor voltage: 80 V); for 3HAA m/z 154.0 (scan period: 12-18 min, fragmentor voltage: 80 V); for 3NT m/z 227.0 (scan period: 18-23 min, fragmentor voltage: 100 V); for XA m/z 206.0 (scan period: 23-45 min, fragmentor voltage: 100 V).</li>
	</ol>
	</li>
	<li>Construct a worklist and run samples on the LC system.</li>
	<li>At first, before the analysis of the experimental samples, run a blank sample (Solvent A) at least in triplicates, followed by one QC sample.</li>
	<li>Open the data analysis software and load the results obtained for the QC sample.</li>
	<li>Check the position of analyte peaks on the chromatogram. When signals shift beyond the expected position adjust the time gates for appropriate analyte signals collection. See the software manual for instruction on signal integration.</li>
</ol>
6. Constructing the calibration curve
<ol>
	<li>In the data acquisition software, add calibration standards into the worklist and run the standards in triplicates.</li>
	<li>Integrate and measure the peak area corresponding to 3HKyn, Kyn, 3HAA, 3NT, and XA at the retention time of about 4.4 min, 10 min, 16 min, 21 min, and 30 min, respectively.</li>
	<li>Construct individual calibration curves for each metabolite using a spreadsheet program.
	<ol>
		<li>To create the calibration chart, plot the value for a ratio of analyte peak area over the 3NT peak area versus the concentration of the analyte.</li>
		<li>Analyze the blank sample (charcoal-pretreated culture medium spiked only with 3NT) to ensure there is no trace of the studied analytes in the medium. Otherwise, subtract the obtained value from each calibration point.</li>
		<li>Check the linearity of each calibration curve.
		<ol>
			<li>Individually, for each analyte, create a slope-intercept linear equation (y = ax +b), where y corresponds to the ratio of the analyte peak area versus 3NT peak area, a is the slope, x is the analyte concentration (&#181;mol/L), and b refers to the curve intercept. Plotting the graph &#8216;y&#8217; versus &#8216;x&#8217; will generate the calibration curve.</li>
			<li>Add a linear trendline to the chart, display the calibration curve equation and regression coefficient on the chart. Ensure that the regression coefficient is &#62; 0.990. In case of mismatched calibration standards, prepare and/or analyze these once again. Optionally, change the concentration range of the calibration curve.</li>
		</ol>
		</li>
		<li>Analyze the QC samples to check the system performance before analyzing the experimental samples. In case of incompatibilities (&#177; 20% deviation from the reference value), prepare the new calibration curves.</li>
	</ol>
	</li>
</ol>
7. In vitro cell culture and sample collection for analysis
<ol>
	<li>Plate the studied cancer cells (MDA-MB-231 or SK-OV-3) in DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium) containing 4.5 g/L of &#7429;-glucose, 10% (v/v) fetal bovine serum (FBS), 2 mmol/L &#671;-glutamine and 1 U penicillin/streptomycin and culture at 37 &#176;C in a humidified atmosphere of 5% CO2 to expand them for the experiment. Passage the cells at 80% of confluency.</li>
	<li>Detach cells from the culture dish using trypsin, seed for the experiment on 12-well plates at a density of 0.3 &#215; 106 cells per well and place in an incubator overnight.</li>
	<li>The next day, aspirate the culture medium and add fresh DMEM with or without the addition of the studied compounds, depending on the experimental design.</li>
	<li>After 48 h, collect the medium from above the cells (about 500 &#181;L) into a 1.5 mL tube. Centrifuge at 14, 000 x g for 5 min to remove any cell debris. Collect the supernatant and store at -80 &#176;C until the analysis.</li>
	<li>Save the cellular pellet from step 7.4 for protein estimation. Freeze the samples at -20 &#176;C. 
	NOTE: The results obtained for a medium from different wells should be normalized to total protein content to account for the variability in cell number in individual wells. Protein concentration can be assessed as described in step 9.</li>
</ol>
8. Prepare samples for LC-MS analysis
<ol>
	<li>Thaw the frozen sample at room temperature and mix well. Transfer 149.25 &#181;L of the culture medium into a centrifugation tube (1.5 mL).</li>
	<li>Add 0.75 &#181;L of 0.1 g/L of 3NT solution (internal standard). Close the tubes and vortex well.</li>
	<li>Add 150 &#181;L of chilled at -20 &#176;C methanol containing 1% (v/v) FA for sample deproteinization. Close the tubes and vortex well.</li>
	<li>Continue with sample preparation as described in section 3 (steps 3.3-3.11). 
	NOTE: Some cancer cells like SK-OV-3 secrete large amounts of Kyn into a culture medium. To fit the data into a linear range of the calibration curve, approximately 100-fold dilution of the sample is necessary. In this case, dilute a portion of the sample with 0.1% (v/v) FA in water and analyze in addition to the undiluted sample. The reference samples of standards for the calibration curve should be prepared in 100 times diluted complete culture medium. Alternatively, add more points in the calibration curve (if proper solubility and linearity are achieved) to adjust it to the concentration level of Kyn in the experimental sample.</li>
	<li>Analyze the samples by LC-MS as described in section 5. Construct a worklist and run each sample in triplicate.</li>
	<li>After the worklist is completed, measure the peak area of analyte.</li>
	<li>Generate a spreadsheet using the available software and the obtain numerical data.</li>
	<li>In one spreadsheet column calculate the ratio of the analyte peak area over the 3NT peak area.</li>
	<li>Use an individual linear calibration equation dedicated for each analyte (from step 6.3.) to calculate concentrations of analytes present in the experimental samples.</li>
</ol>
9. Assessing protein content and data normalization
<ol>
	<li>Resuspend the cellular pellet from step 7.5 with 100 &#181;L of phosphate-buffered saline (PBS) in 1.5 mL tube. 
	NOTE: Prepare PBS solution by dissolving 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium phosphate dibasic, potassium dihydrogen phosphate in 950 mL of ultrapure water. Stir well. Adjust the pH to 7.4 with hydrochloric acid (dropwise). Adjust volume up to 1 L with ultrapure water. Stir well until homogeneous. 
	CAUTION: Hydrochloric acid is highly corrosive and must be handled with suitable safety precautions. Contact with human skin can cause redness, pain, skin burns. Work under the fume hood; wear protective gloves and coat.</li>
	<li>Freeze the samples at &#160;-20 &#176;C and then defrost on ice. Repeat this step 3x.</li>
	<li>Centrifuge the samples at 14, 000 x g for 15 min at 4 &#176;C. Collect the supernatants into the new tubes. Do not disturb the pellet.</li>
	<li>Dilute the supernatants 10x with PBS.</li>
	<li>Construct a calibration curve from 0 to 2&#160;&#181;g of the protein per well range.
	<ol>
		<li>Prepare bovine serum albumin (BSA) standard solution for the calibration. Dissolve 1.23 &#181;g of BSA in 1 mL of ultrapure water and vortex well.</li>
		<li>On a 96-well plate, load different amounts of BSA standard solution (1.23 &#181;g/mL): 0 &#181;L, 2 &#181;L, 4 &#181;L, 5 &#181;L, 7 &#181;L, 10 &#181;L, 15 &#181;L, 20 &#181;L.</li>
		<li>Fill the well with PBS to the final volume of 50 &#181;L.</li>
	</ol>
	</li>
	<li>Load 10 - 15 &#181;L cell lysate from step 9.4&#160;on the same 96-well plate. Fill the wells with PBS to reach to the final volume of 50 &#181;L. 
	NOTE: If needed, adjust the volume of the cell lysate aliquot in each well to fit in the linear range of the calibration curve. Keep the final volume to 50 &#181;L of the sample per well.</li>
	<li>Add 200 &#181;L of a Bradford reagent (diluted 5-times with ultrapure water) to each well.</li>
	<li>Incubate the 96-well pate for at least 5 min at room temperature. Insert the plate into a microplate reader. Measure absorbance at &#955; = 595 nm.</li>
	<li>Construct a calibration curve using a spreadsheet program. Plot the BSA amount (&#181;g) versus absorbance. Add a linear trendline, display the calibration curve equation and regression coefficient on the chart.</li>
	<li>Use the linear equation (y = ax +b, where y corresponds to the absorbance at &#955; = 595 nm, a is the slope, x is a protein content (&#181;g), and b refers to the curve intercept) to calculate protein amount in the sample. 
	NOTE: Consider a sample dilution factor in calculations.</li>
	<li>Use the estimated protein content to normalize kynurenine amount in the sample per 1 mg of protein. To do this, divide the concentration of kynurenines determined in step 8.9 with the total protein content from step 9.10.</li>
</ol>

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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>

The authors have nothing to disclose.

This paper presents a detailed LC-SQ protocol for the simultaneous quantification of four major Trp metabolites (3HKyn, Kyn, 3HAA, XA) that are measured in a medium from in vitro cultured human cancer cells. Special attention is paid to the sample preparation, chromatographic/mass spectrometry procedure, and data interpretation, the most important points within the analysis.
In general, LC-MS analysis, due to high sensitivity, requires the highest standards of a protocol strictness an...

Kynurenine pathway (KP) is the major route of tryptophan (Trp) catabolism in human cells. Indoleamine-2,3-dioxygenase (IDO-1) in extrahepatic cells is the first and limiting enzyme of KP and converts Trp into N-formylkynurenine1. Further steps within KP generate other secondary metabolites, namely kynurenines that exhibit various biological properties. Kynurenine (Kyn) is the first stable Trp catabolite showing toxic properties and regulating cellular events after binding to the aryl hydrocarbon receptor (AhR)2. Subsequently, Kyn is transformed into several molecules either spontaneously or in the enzyme-mediated processes, generating such metabolites like 3-hydroxykynurenine (3HKyn), anthranilic acid (AA), 3-hydroxyanthranilic acid (3-HAA), kynurenic acid (Kyna), and xanthurenic acid (XA). Another downstream metabolite, 2-amino-3-carboxymuconic acid-6-semialdehyde (ACMS), undergoes non-enzymatic cyclization to quinolinic acid (QA) or picolinic acid (PA)1. Finally, QA is further transformed into nicotinamide-adenine dinucleotide (NAD+)3, the KP end-point metabolite that is an important enzyme cofactor. Some kynurenines have neuroprotective properties such as Kyna and PA, while the others, i.e., 3HAA and 3HKyn, are toxic4. Xanthurenic acid, which is formed from 3HKyn, presents antioxidant and vasorelaxation properties5. XA accumulates in aging lenses and leads to apoptosis of epithelial cells6. KP, described in the middle of the 20th century, gained more attention when its involvement in various disorders was demonstrated. Increased activity of this metabolic route and accumulation of some kynurenines modulate the immune response and are associated with different pathological conditions such as depression, schizophrenia, encephalopathy, HIV, dementia, amyotrophic lateral sclerosis, malaria, Alzheimer&#8217;s, Huntington&#8217;s disease, and cancer4,7. Some changes in Trp metabolism are observed in tumor microenvironments and cancer cells2,8. Moreover, kynurenines are considered as promising disease markers9. In cancer research, in vitro cell culture models are well established and widely used for preclinical evaluation of responses to anticancer drugs10. Trp metabolites are secreted by cells into the culture medium and can be measured to assess the status of the kynurenine pathway. Therefore, there is a need to develop appropriate methods for the simultaneous detection of as many KP metabolites as possible in a variety of biological specimens with an easy, flexible, and reliable protocol.
In this paper, we describe a protocol for the simultaneous determination of four kynurenine pathway metabolites: Kyn, 3HKyn, 3HAA, and XA by LC-SQ&#160;in a post-culture medium collected from cancer cells. In a modern analytical approach, liquid chromatography13,14,15,16 is preferred for the simultaneous detection and quantification of the individual tryptophan catabolites, in contrast to biochemical nonspecific assays utilizing Ehrlich reagent11,12. At present, there are many methods available for kynurenines determination in human specimens, &#160;mainly based on liquid chromatography with ultraviolet or fluorescence detectors13,17,18,19. Liquid chromatography coupled with a mass spectrometry detector (LC-MS) seems more suitable for this type of analysis, due to their higher sensitivity (lower limits of detection), selectivity and repeatability. &#160;
Trp metabolites have already been determined in human serum, plasma and urine13,20,21,22,23, however, the methods for other biological specimens, like cell culture medium are also desired. Previously, LC-MS was used for Trp-derived compounds in a medium collected after culturing of human glioma cells, monocytes, dendritic cells or astrocytes treated with interferon gamma (IFN-&#947;)24,25,26. Currently, there is a need for new validated protocols that can allow an assessment of several metabolites in different culture media, cells, and treatments used in cancer models.
The purpose of the developed&#160;method is to quantify (within one analytical run) four major kynurenines that can indicate abnormalities in KP. Presented here are critical steps of our recently published protocol for quantitative LC-SQ analysis of selected meaningful kynurenines using one internal standard (3-nitrotyrosine, 3NT) in the medium collected from in vitro cultured human cancer cells27. To our best knowledge, it is the first LC-SQ protocol for simultaneous quantification of 3HKyn, 3HAA, Kyn and XA in a culture medium obtained from the in vitro grown cells. Upon some modifications, the method might be further applied to study the changes in Trp metabolism in a broader range of cell culture models.

<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

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

LC-MS presents indisputable advantages in the quantification of biologically active molecules, even though some components of complex specimens cause so-called matrix effects and compromise ionization of analytes. It leads to ion suppression or ion enhancement, strongly decreasing accuracy and affecting a limit of detection/quantification of LC-MS, which is considered as a &#39;&#39;weak&#8221; point of the method. In our protocol, the ions are generated by electrospray ionization (ESI) in the positive mode, which was al...

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

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

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 Views.  The John Paul II Catholic University of Lublin. Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Video: 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).

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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