This work was supported by the National Institutes of Health grant R35GM119528 to Roberta Leonardi.

All experimental procedures on mice (C57BL/6J, males, 9-11 weeks of age) were approved by the Institutional Animal Care and Use Committees (IACUC) of West Virginia University.
1. Hepatocyte isolation
<ol>
	<li>Preparation
	<ol>
		<li>In the days before the hepatocyte isolation, prepare the buffers and cell culture media listed in Table 1. Set up a water bath with the temperature set to 37 &#176;C close to where the surgery will be performed.</li>
		<li>On the day of the hepatocyte isolation, under a laminar flow hood, transfer 35 mL of Buffer 1 to a sterile 50 mL centrifuge tube and 70 mL of Buffer 2 to a 100 mL sterile beaker or bottle.</li>
		<li>Add antibiotics gentamicin (50 &#181;g/mL) and penicillin/streptomycin (1x) to both buffers.</li>
		<li>Transfer 20 mL of Buffer 2 as prepared in step 1.1.3 to a 100 mm cell culture dish and place on ice.</li>
		<li>Transfer the remaining 50 mL of Buffer 2 to a sterile 50 mL centrifuge tube. Place the 50 mL tubes containing the antibiotic-supplemented Buffers 1 and 2 in a water bath set at 37 &#176;C and let them warm up for at least 15 min before starting the perfusion. 
		NOTE: If conducting multiple hepatocytes isolations in a session, scale up the number of antibiotic-supplemented aliquots of Buffers 1 and 2 to prepare accordingly.</li>
		<li>Thaw an aliquot of collagenase solution and keep on ice. 
		NOTE: If properly stored, there is no significant loss of enzyme activity in collagenase solutions frozen and thawed up to 3 times and used within 3 weeks from preparation.</li>
		<li>Prepare the surgical instruments and peristaltic pump. Sterilize the lines of the peristaltic pump by circulating 15 mL of 70% ethanol, followed by 15 mL of sterile water.</li>
		<li>Connect a 22 G needle to the exit line (Figure 1A). The hollowed filter of a catheter works well as a connector. Fill the lines with Buffer 1 and inspect the lines, connector, and needle to ensure no air bubbles are trapped.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62904/62904fig1v2.jpg" /> 
Figure 1: Perfusion apparatus and perfused liver. (A) Peristaltic pump with outlet line connected to the needle used to cannulate and perfuse the liver. (B) Successful cannulation is indicated by immediate and homogeneous blanching of the liver. <a href="https://www.jove.com/files/ftp_upload/62904/62904fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Liver perfusion and dissociation
	<ol>
		<li>Anesthetize a mouse via isoflurane inhalation with medical grade air as carrier gas, using 4% isoflurane for induction and 1.5% isoflurane to maintain anesthesia. Verify the depth of anesthesia by assessing the loss of pedal reflex.</li>
		<li>When there is no response to toe pinching, place the mouse in a supine position on a surgery board, outstretch the limbs, and secure them to the board with pins.</li>
		<li>Liberally spray the abdomen and chest of the mouse with 70% ethanol.</li>
		<li>Using forceps, pull up the skin and abdominal wall near the base of the abdomen and cut laterally, on either side of the midline and up to the diaphragm, to expose the organs.</li>
		<li>Expose the inferior vena cava (IVC) by moving the intestines to the right side and gently flipping the lobes of the liver up. Insert a small cylindrical object, such as a needle cap, under the back of the mouse to slightly tilt the IVC and facilitate its cannulation (Figure 1B).</li>
		<li>Start the pump at the lowest speed and, with Buffer 1 flowing, insert the needle into the IVC.</li>
		<li>Cut the portal vein to relieve the pressure and allow for drainage of blood and perfusion buffers, then immediately increase the flow rate to 7 mL/min. If done correctly, the liver will uniformly blanch within a few seconds (Figure 1B).</li>
		<li>For more consistent results, hold the needle in position by hand for the entire duration of the perfusion.</li>
		<li>Perfuse the liver with warm Buffer 1. To avoid introducing air bubbles, ensure that the line inserted in the tube containing Buffer 1 remains continuously submerged.</li>
		<li>While perfusion occurs, add 130 &#181;L of collagenase solution to Buffer 2 and mix by pipetting up and down or stirring with a 5 mL or 10 mL serological pipette.</li>
		<li>As the volume in the tube containing Buffer 1 decreases to about 5 mL, slowly add 5 mL of Buffer 2 to Buffer 1 by pipetting on the side of the tube. The goal is to avoid introducing air bubbles in the line while changing from Buffer 1 to Buffer 2.</li>
		<li>Wait until the volume decreases again to 5 mL and slowly add another 5 mL of Buffer 2. Repeat one more time. As Buffer 2 replaces Buffer 1 and the dissociation starts, the liver will swell.</li>
		<li>Add the remaining Buffer 2 to the tube originally containing Buffer 1. Stop the perfusion when there is about 5-10 mL of Buffer 2 left in the tube. 
		NOTE: While Buffer 2 is perfusing the liver, the portal vein can be intermittently clamped with forceps for 5 s. This step is optional, but the resultant increase in pressure throughout the liver can improve its dissociation and, thus, the final hepatocyte yield.</li>
		<li>Carefully excise the liver and transfer it to the 100 mm culture dish containing the 20 mL of ice-cold Buffer 2 set aside at step 1.1.4.</li>
		<li>Under the laminar flow hood, gently break the liver apart using surgical scissors and tweezers.</li>
		<li>Add about 20 mL of ice-cold M199 to the hepatocyte suspension and filter it through a 100 &#181;m cell strainer using the plunger of a syringe to gently promote the release of additional hepatocytes from larger liver pieces.</li>
		<li>Wash the 100 mm culture dish and the cell strainer with additional M199 until the collection tube is full.</li>
		<li>Centrifuge the suspension at 50 x g for 2 min at 4 &#176;C. Carefully aspirate the supernatant and gently resuspend the hepatocyte pellet in 30 mL of cold M199 by swirling.</li>
		<li>Pellet the hepatocytes as mentioned in step 1.2.18. Repeat the wash one more time.</li>
		<li>Resuspend the hepatocytes in 10 mL of warm M199 and determine the viability and yield using the trypan blue exclusion method and a hemocytometer12.</li>
		<li>Dilute the cells in M199 warmed to 37&#160;&#186;C to a final concentration of 1.0 x 106 viable cells/mL and immediately start the assay.</li>
	</ol>
	</li>
</ol>
2. Fatty acid &#946;-oxidation assay
NOTE: The assay is conducted in triplicate, and each reaction mixture contains 750,000 cells, 1.35 mg/mL bovine serum albumin (BSA), 100 &#181;M palmitic acid, and 0.4 &#181;Ci [1-14C]palmitic acid in a final volume of 2 mL. 
CAUTION: Radioactive compounds are hazardous. Purchase, handle, store, and dispose of radioactive material in accordance with Institutional, State, and Federal regulations.
<ol>
	<li>Preparation
	<ol>
		<li>In the days before the assay, prepare the palmitic acid and BSA solutions (Table 1) and store them at -20 &#176;C.</li>
		<li>On the day of the assay, complete steps 2.1.3-2.1.9 before starting the liver perfusion.</li>
		<li>Thaw the palmitic acid and BSA solutions. Prepare the substrate mixture for multiple reactions plus a 20%-30% excess, with a typical assay setup shown in Table 2.</li>
		<li>Aliquot 13.5 &#181;L of BSA solution per reaction in a microcentrifuge tube and warm to 41 &#176;C, then add 1 &#181;L of the 200 mM palmitic acid solution (BSA: palmitic acid molar ratio = 1:5) per reaction. 
		NOTE: It is preferable to dispense solutions prepared using organic solvents, such the radioactive and non-radioactive palmitic acid solutions, with a positive displacement pipette and appropriate tips.</li>
		<li>Vortex vigorously and incubate at 41 &#176;C to facilitate the formation of the soluble palmitic acid: BSA complex. Vortex occasionally during the incubation period.</li>
		<li>The mixture will initially appear cloudy but will clarify completely after 20-30 min of incubation at 41 &#176;C. Keep it at 41 &#176;C until ready to start the reactions.</li>
		<li>Aliquot 133 &#181;L of 1 M perchloric acid in 1.5 mL microcentrifuge tubes to stop the reactions. 
		CAUTION: Perchloric acid is a strong acid and a strong oxidant. Appropriate protective gear is required for handling this compound.</li>
		<li>Aliquot 485.5 &#181;L of M199 per reaction into a tube and keep it at 37 &#176;C to dilute the radioactive BSA: palmitic acid complex prepared at steps 2.1.4-2.1.6&#160;before starting the reactions.</li>
		<li>Dispense 750 &#181;L of M199 in as many 14 mL round-bottom tubes as the samples. If desired, add inhibitors of fatty acid &#946;-oxidation, such as etomoxir, rotenone, and antimycin, including a vehicle control (Table 2).</li>
		<li>During the hepatocyte wash steps, 10-15 min before starting the reactions, transfer the tubes prepared at step 2.1.9&#160;to a shaking water bath set to 37 &#176;C and shaking at 180-200 rpm.</li>
	</ol>
	</li>
	<li>Starting, stopping, and analyzing the fatty acid &#946;-oxidation reactions
	<ol>
		<li>If the viability of the hepatocytes is acceptable (typically &#8805; 75%, Figure 2), for each reaction, transfer 0.8 &#181;L of [1-14C]palmitic acid (0.5 mCi/mL) to the microcentrifuge tube containing the clarified BSA: palmitic acid solution (steps 2.1.4-2.1.6). Vortex and return to the water bath at 41 &#176;C.</li>
		<li>To equilibrate the hepatocytes to 37 &#176;C and to pre-incubate them with inhibitors (if present), immediately after the final hepatocyte resuspension (step 1.2.21), transfer 750 &#181;L of the hepatocyte suspension with a 1 mL pipette to each of the 14 mL round-bottom tubes in the shaking water bath (steps 2.1.9-2.1.10) and vortex briefly at low speed to mix.</li>
		<li>Stagger each addition by 30 s and incubate for 15 min. To save a sample for protein determination, transfer another aliquot of hepatocytes to a 1.5 mL microcentrifuge tube and spin at 3,000 x g for 5 min. 
		NOTE: While dispensing, the hepatocyte suspension needs to be continuously swirled or gently stirred with the dispensing 1 mL pipette to prevent settling and large variability in cell number across samples.</li>
		<li>Remove the supernatant and store the pellet at -80 &#176;C until ready to measure the total amount of protein in the sample to normalize the results (Figure 3).</li>
		<li>While the hepatocytes are under pre-incubation at 37 &#176;C, add the radioactive BSA: palmitic acid complex to the warm medium in 2.1.8, and keep at 37 &#176;C until ready to start the reactions. This is the final substrate mix.</li>
		<li>To start the reactions, remove the hepatocytes from the water bath and add 500 &#181;L of substrate mix.</li>
		<li>Vortex at low speed for 5 s to completely resuspend the cells and return to the water bath. Repeat with all the samples, staggering by 30 s.</li>
		<li>Incubate for 15 min. Start a set of reactions and immediately stop (see steps 2.2.10-2.2.11) to determine the background radioactivity (Table 2).</li>
		<li>Transfer duplicate aliquots (200-250 &#181;L) of the leftover substrate mix to 6 mL scintillation vials and set aside to count. Use these counts to calculate the radioactivity corresponding to the total nmoles of palmitic acid available for oxidation in 500 &#181;L of substrate mix.</li>
		<li>To stop the reactions, remove the hepatocytes from the water bath, resuspend the hepatocytes by vortexing at moderate speed, and then transfer 400 &#181;L of the hepatocyte suspension to the microcentrifuge tubes containing perchloric acid.</li>
		<li>Immediately cap the tubes. Repeat this sequence for all the samples, staggering by 30 s.</li>
		<li>Vigorously vortex the 1.5 mL microcentrifuge tubes and spin them&#160;down the 1.5 mL microcentrifuge tubes at 13,000 x g for 10 min.</li>
		<li>Transfer 300 &#181;L of the supernatant to a 6 mL scintillation vial, add 4 mL of scintillation fluid, and count the radioactivity in the samples and the substrate mix aliquots (step 2.2.9) in a scintillation counter. 
		CAUTION: After centrifuging, open the tubes under a fume hood to avoid breathing the 14C-CO2 produced by the complete oxidation of 14C-acetyl-CoA generated by fatty acid &#946;-oxidation and released by the acidic conditions.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffers/Media Components</td>
			<td>Amount&#160;</td>
			<td>Final Concentration</td>
			<td>Instructions</td>
		</tr>
		<tr>
			<td colspan="4">Solution C</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&#160;1.79 g</td>
			<td>480 mM</td>
			<td rowspan="3">Add water to 50 mL.&#160; Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>MgSO4 heptahydrate</td>
			<td>&#160;1.48 g</td>
			<td>120 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>&#160;0.81 g</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td colspan="4">Krebs-Henseleit Buffer (KHB), calcium-free</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>7.0 g</td>
			<td>&#160;120 mM</td>
			<td rowspan="5">Add water to 900 mL, adjust the pH to 7.4, and bring the final volume to 1 L. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>2.0 g</td>
			<td>&#160;24 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES pH 7.45</td>
			<td>5 mL</td>
			<td>&#160;5 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1 or 2 g</td>
			<td>&#160;5.6 or 11 mM</td>
		</tr>
		<tr>
			<td>Solution C</td>
			<td>10 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Buffer 1</td>
		</tr>
		<tr>
			<td>KHB</td>
			<td>500 mL</td>
			<td></td>
			<td rowspan="2">Mix components and filter sterilize. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>50 mM EGTA</td>
			<td>1.0 mL</td>
			<td>&#160;0.1 mM</td>
		</tr>
		<tr>
			<td colspan="4">Buffer 2</td>
		</tr>
		<tr>
			<td>KHB</td>
			<td>500 mL</td>
			<td></td>
			<td rowspan="2">Mix components and filter sterilize. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>1 M CaCl2 dihydrate</td>
			<td>686 &#181;L</td>
			<td>&#160;1.4 mM</td>
		</tr>
		<tr>
			<td colspan="4">Gentamicin solution</td>
		</tr>
		<tr>
			<td>Gentamicin sulphate</td>
			<td>0.5 g</td>
			<td>50 mg/mL</td>
			<td>Add water to 10 mL and filter sterilize. Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">Collagenase solution</td>
		</tr>
		<tr>
			<td>Collagenase I and II blend</td>
			<td>10 mg</td>
			<td>7 mg/mL</td>
			<td>Dissolve the entire content of the vial in 1.43 mL of water.&#160; Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">M199</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>1 pouch</td>
			<td></td>
			<td rowspan="4">Add water to 900 mL and adjust the pH to 7.2-7.4. Bring the final volume to&#160; 1 L and filter sterilize. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>2.2 g</td>
			<td>&#160;26 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES (cell culture grade)</td>
			<td>25 mL</td>
			<td>&#160;25 mM</td>
		</tr>
		<tr>
			<td>Extra glucose (only for fed mice)</td>
			<td>1 g</td>
			<td>&#160;11 mM</td>
		</tr>
		<tr>
			<td colspan="4">BSA solution</td>
		</tr>
		<tr>
			<td>Fatty acid-free BSA</td>
			<td>400 mg</td>
			<td>20% (w/v)</td>
			<td>Dissolve in 2 mL of water.&#160; Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">Non-radioactive palmitic acid solution</td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>103 mg</td>
			<td>200 mM</td>
			<td>Dissolve in 2 mL of ethanol, store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">1 M Perchloric acid</td>
		</tr>
		<tr>
			<td>70% Perchloric acid</td>
			<td>3.5 mL</td>
			<td>1 M</td>
			<td>Dilute to 40 mL with water. Store at room temperature&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers, media, and other solutions required for the hepatocyte isolation and the fatty acid &#946;-oxidation assay
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Reaction number</td>
			<td colspan="2">M199 &#177; Inhibitors</td>
			<td></td>
			<td rowspan="2">Hepatocyte suspension (&#181;L)</td>
			<td></td>
			<td rowspan="2">Substrate mix (&#181;L)</td>
			<td></td>
		</tr>
		<tr>
			<td>Volume (&#181;L)</td>
			<td>Etomoxir</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1</td>
			<td rowspan="9">750</td>
			<td rowspan="3">-</td>
			<td rowspan="9">Pre-warm at 37 &#176;C</td>
			<td rowspan="9">750</td>
			<td rowspan="9">Pre-incubate at 37 &#176;C for 15 min</td>
			<td rowspan="9">500</td>
			<td rowspan="6">Incubate at 37 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td>2</td>
		</tr>
		<tr>
			<td>3</td>
		</tr>
		<tr>
			<td>4</td>
			<td rowspan="3">+</td>
		</tr>
		<tr>
			<td>5</td>
		</tr>
		<tr>
			<td>6</td>
		</tr>
		<tr>
			<td>7</td>
			<td rowspan="3">+</td>
			<td rowspan="3">Stop immediately</td>
		</tr>
		<tr>
			<td>8</td>
		</tr>
		<tr>
			<td>9</td>
		</tr>
	</tbody>
</table>
Table 2: Example of the experimental setup for a hepatocyte suspension assayed in triplicate in the presence and absence of etomoxir.

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(79), e50615 (2013).</li><li>Lilly, K., Chung, C., Kerner, J., VanRenterghem, R., Bieber, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+etomoxiryl-CoA+on+different+carnitine+acyltransferases.">Effect of etomoxiryl-CoA on different carnitine acyltransferases.</a> Biochemical Pharmacology. 43 (2), 353-361 (1992).</li><li>Yu, X. X., Drackley, J. K., Odle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+mitochondrial+and+peroxisomal+beta-oxidation+of+palmitate+change+during+postnatal+development+and+food+deprivation+in+liver,+kidney+and+heart+of+pigs.">Rates of mitochondrial and peroxisomal beta-oxidation of palmitate change during postnatal development and food deprivation in liver, kidney and heart of pigs.</a> Journal of Nutrition. 127 (9), 1814-1821 (1997).</li><li>Yu, X. X., Drackley, J. K., Odle, J., Lin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+hepatic+mitochondrial+and+peroxisomal+beta-oxidation+to+increasing+palmitate+concentrations+in+piglets.">Response of hepatic mitochondrial and peroxisomal beta-oxidation to increasing palmitate concentrations in piglets.</a> Biology of the Neonate. 72 (5), 284-292 (1997).</li><li>Veerkamp, J. H., van Moerkerk, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+fatty+acid+oxidation+in+rat+and+human+tissues.+Effect+of+nutritional+state,+clofibrate+treatment+and+postnatal+development+in+the+rat.">Peroxisomal fatty acid oxidation in rat and human tissues. Effect of nutritional state, clofibrate treatment and postnatal development in the rat.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 875 (2), 301-310 (1986).</li><li>Hakvoort, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interorgan+coordination+of+the+murine+adaptive+response+to+fasting.">Interorgan coordination of the murine adaptive response to fasting.</a> Journal of Biological Chemistry. 286 (18), 16332-16343 (2011).</li><li>Sokolovic, 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=The+transcriptomic+signature+of+fasting+murine+liver.">The transcriptomic signature of fasting murine liver.</a> BMC Genomics. 9, 528 (2008).</li><li>Kersten, 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=Peroxisome+proliferator-activated+receptor+alpha+mediates+the+adaptive+response+to+fasting.">Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.</a> Journal of Clinical Investigation. 103 (11), 1489-1498 (1999).</li><li>Li, W. C., Ralphs, K. L., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+hepatocytes.">Isolation and culture of adult mouse hepatocytes.</a> Methods in Molecular Biology. 633, 185-196 (2010).</li><li>Ng, I. 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=Isolation+of+Primary+Rat+Hepatocytes+with+Multiparameter+Perfusion+Control.">Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control.</a> Journal of Visualized Experiments: JoVE. (170), e62289 (2021).</li><li>Shen, L., Hillebrand, A., Wang, D. Q., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+hepatic+cells.">Isolation and primary culture of rat hepatic cells.</a> Journal of Visualized Experiments: JoVE. (64), e3917 (2012).</li><li>Fulgencio, J. P., Kohl, C., Girard, J., Pegorier, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+metformin+on+fatty+acid+and+glucose+metabolism+in+freshly+isolated+hepatocytes+and+on+specific+gene+expression+in+cultured+hepatocytes.">Effect of metformin on fatty acid and glucose metabolism in freshly isolated hepatocytes and on specific gene expression in cultured hepatocytes.</a> Biochemical Pharmacology. 62 (4), 439-446 (2001).</li><li>Leonardi, R., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pank1+deletion+in+leptin-deficient+mice+reduces+hyperglycaemia+and+hyperinsulinaemia+and+modifies+global+metabolism+without+affecting+insulin+resistance.">Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.</a> Diabetologia. 57 (7), 1466-1475 (2014).</li><li>Bougarne, N., 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=PPARalpha+blocks+glucocorticoid+receptor+alpha-mediated+transactivation+but+cooperates+with+the+activated+glucocorticoid+receptor+alpha+for+transrepression+on+NF-kappaB.">PPARalpha blocks glucocorticoid receptor alpha-mediated transactivation but cooperates with the activated glucocorticoid receptor alpha for transrepression on NF-kappaB.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (18), 7397-7402 (2009).</li><li>Korelova, K., Jirouskova, M., Sarnova, L., Gregor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+3D+collagen+sandwich+culture+of+primary+mouse+hepatocytes+to+study+the+role+of+cytoskeleton+in+bile+canalicular+formation+in+vitro.">Isolation and 3D collagen sandwich culture of primary mouse hepatocytes to study the role of cytoskeleton in bile canalicular formation in vitro.</a> Journal of Visualized Experiments: JoVE. (154), e60507 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

During the liver perfusion, it is critical to avoid the introduction of air bubbles, as they block the microcapillaries in the liver, preventing or restricting the buffer circulation and overall decreasing the hepatocyte yield and viability20,21. Precautions, such as closely inspecting the buffer-filled inlet line before cannulation of the IVC and avoiding lifting the inlet line off the tube containing Buffer 1 to switch to Buffer 2, as described herein, can succ...

Fatty acid &#946;-oxidation is an essential process in lipid metabolism, providing a catabolic pathway to balance fatty acid synthesis and intake from the diet. This process generates energy for multiple organs, including the cardiac muscle, kidney cortex, and fasted liver, and utilizes fatty acids obtained from the diet, adipose tissue lipolysis, and internal triglyceride stores1,2.
Oxidation of fatty acid through the &#946;-oxidation pathway results in the sequential shortening of the fatty acyl chain by two carbons at a time, released as acetyl-CoA, and this process occurs both in the mitochondria and the peroxisomes. While most fatty acids undergo only &#946;-oxidation, some are oxidized at different carbons before entering this pathway. For example, 3-methyl-substituted fatty acids, such as phytanic acid, undergo removal of one carbon by &#945;-oxidation in the peroxisomes before entering the &#946;-oxidation pathway. Similarly, some fatty acids are first converted to dicarboxylic fatty acids by oxidation of the terminal methyl group (&#969;-oxidation) in the endoplasmic reticulum before being preferentially oxidized in the peroxisomes by &#946;-oxidation3.
Regardless of the specific organelle, a fatty acid must first be converted to a coenzyme A (CoA) thioester, or acyl-CoA, to be oxidized through the &#946;-oxidation pathway. &#946;-Oxidation of long-chain acyl-CoAs in the mitochondrial matrix requires the carnitine shuttle for their translocation, where carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of acyl-CoAs to acylcarnitines and is the rate-limiting enzyme in this process4. Once translocated to the mitochondrial matrix, the acyl-CoAs are re-formed and serve as substrates for the mitochondrial &#946;-oxidation machinery. In the fasted state, the acetyl-CoA produced through &#946;-oxidation in hepatic mitochondria is primarily channeled to ketogenesis. Peroxisomes serve as the primary site for the &#946;-oxidation of very long-chain, branched-chain, and dicarboxylic fatty acids. Peroxisomes do not require the carnitine shuttle to import fatty acid substrates, instead&#160;importing the correspondent acyl-CoAs through the activity of the ATP-binding cassette (ABC) transporters ABCD1-35. Within the peroxisomes, acyl-CoAs are then oxidized by a dedicated set of enzymes, distinct from the mitochondrial fatty acid &#946;-oxidation machinery. Both mitochondria and peroxisomes also require a supply of NAD+ and free CoA to oxidize fatty acyl chains. CoA levels in the liver have been shown to increase in response to fasting, supporting the increased rate of fatty acid oxidation which occurs in this state6. Furthermore, increased CoA degradation in the peroxisomes results in a selective decrease in peroxisomal fatty acid oxidation7. Therefore, the process of fatty acid oxidation within the cell is regulated by the expression levels and activities of enzymes involved in the activation, transport, and oxidation of fatty acids, as well as the concentrations of cofactors and other metabolites throughout multiple subcellular compartments.
Procedures using tissue homogenates to measure fatty acid oxidation destroy the cellular architecture regulating and supporting this process, leading to a collection of data that does not accurately reflect the in vivo metabolism. While techniques using plated primary hepatocytes preserve this system, culturing isolated cells for extended periods of time results in a loss of the in vivo gene expression profile that was present in the cells when they were still living within the animal8,9. The following protocol describes a method to isolate primary hepatocytes and assay their capacity for fatty acid &#946;-oxidation immediately after isolation and in suspension, using [1-14C]palmitic acid. The assay is based on the measurement of the radioactivity associated with the acid-soluble metabolites (ASM) or products, like acetyl-CoA, produced by the &#946;-oxidation of [1-14C]palmitic acid10,11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(R)-(+)-Etomoxir sodium salt</td>
			<td>Tocris Bioscience</td>
			<td>4539/10</td>
			<td></td>
		</tr>
		<tr>
			<td>[1-14C]-Palmitic acid, 50&#8211;60 mCi/mmol, 0.5 mCi/mL</td>
			<td>American Radiolabeled Chemicals</td>
			<td>ARC 0172A</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES, sterile</td>
			<td>Corning</td>
			<td>25060CI</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L disposable capillaries/pistons for positive displacement pipette</td>
			<td>Mettler Toledo</td>
			<td>17008604</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L, 200 &#181;L, and 10 &#181;L pipettes and tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL, 10 mL, and 25 mL serological pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>CellTreat</td>
			<td>229421</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Perchloric acid</td>
			<td>Fisher Scientific</td>
			<td>A2296-1LB</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, fatty acid-free</td>
			<td>Fisher Scientific</td>
			<td>BP9704100</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 dihydrate</td>
			<td>MilliporeSigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>MilliporeSigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Gold Biotechnology</td>
			<td>E-217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco</td>
			<td>111000200CSPP</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System, 0.22 &#956;m PES Filter, 500 mL, Sterile</td>
			<td>CellTreat</td>
			<td>229707</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulphate</td>
			<td>Gold Biotechnology</td>
			<td>G-400-25</td>
			<td></td>
		</tr>
		<tr>
			<td>HDPE, 6.5 mL scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>03-342-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needles 22 G, 1.5 in</td>
			<td>BD Biosciences</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VetOne</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MilliporeSigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TM Research Grade</td>
			<td>MilliporeSigma</td>
			<td>5401119001</td>
			<td>Defined blend of purified collagenase I and II with a medium concentration of thermolysin</td>
		</tr>
		<tr>
			<td>M199 medium</td>
			<td>MilliporeSigma</td>
			<td>M5017</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 heptahydrate</td>
			<td>MilliporeSigma</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>accuSpin Micro 17</td>
		</tr>
		<tr>
			<td>Microdissecting Scissors</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>RS-5980</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chem-Impex International</td>
			<td>30070</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Acros Organics</td>
			<td>424270010</td>
			<td></td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>MilliporeSigma</td>
			<td>P0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin (100x)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive displacement pipette MR-10, 10 &#181;L</td>
			<td>Mettler Toledo</td>
			<td>17008575</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge with inserts for 50 mL conical tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>5810 R</td>
		</tr>
		<tr>
			<td>Round-bottom, 14 mL, polypropylene culture test tubes</td>
			<td>Fisher Scientific</td>
			<td>14-956-9A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>TriCarb 4810 TR</td>
		</tr>
		<tr>
			<td>ScintiVerse BD cocktail</td>
			<td>Fisher Scientific</td>
			<td>SX18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking water bath, 30 L capacity</td>
			<td>New Brunswick Scientific</td>
			<td></td>
			<td>&#160;Model G76</td>
		</tr>
		<tr>
			<td>Sterile cell strainers, 100 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Thumb Dressing Forceps</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>RS-8120</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>25900CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable-flow peristaltic pump</td>
			<td>Fisher Scientific</td>
			<td>138762</td>
			<td></td>
		</tr>
		<tr>
			<td>Water baths, 2&#8211;2.5 L capacity</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated mouse hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

The liver perfusion described here typically yields 30-40 million cells/liver with average viability of 80%, as estimated by trypan blue exclusion (Figure 2). The typical concentration of glucose in the Krebs-Henseleit buffer (KHB), which is used to prepare the perfusion Buffers 1 and 2, is 11 mM. When measuring fatty acid &#946;-oxidation in hepatocytes isolated from fasted mice, the concentration of glucose in the KHB can be lowered to better represent the fasted state. As shown in <strong...

Watch this Scientific Journal Video about Measurement of Fatty Acid ÃŽÂ²-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes at JoVE.com

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

measurement-fatty-acid-oxidation-suspension-freshly-isolated-mouse

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3.7K Views.  West Virginia University. Fatty acid β-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Video: Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...