Most current bioanalytical methods available for the measurement of bioactive lipids are not as extensive as they do not include chiral normal phase chromatography or SID. Chiral normal phase LC is critical for the separation of enantiomers and for being able to distinguish between enzyme- or ROS-mediated metabolism. The use of SID ensures that human error or complications that arise during extraction or analysis are taken into account during quantification. These added components along with ECAPCI-MRM make this the most sensitive, selective method available for the analysis of bioactive lipids.

1. Standard and Internal Standard Mixes
<ol>
	<li>Before lipid extraction, one must prepare a standard mix (SM) and an internal standard mix (ISM). For the SM, aliquot the equivalent volume of 1 &mu;g of each standard (25 total) listed in the reagent table below, using a calibrated syringe, into a 10 mL volumetric flask. Dry the standards under nitrogen and reconstitute in 10 mL of acetonitrile (ACN). The final concentration of the standard mix will be 100 pg/&mu;L. Divide into 1 mL aliquots and store at -80&deg;C until needed.</li>
	<li>From this standard mix, 6 standard dilutions can be made for a total of 7 calibration standards (1 pg/ &mu;L, 2 pg/ &mu;L, 5 pg/ &mu;L, 10 pg/ &mu;L, 20 pg/ &mu;L, 50 pg/ &mu;L, and 100 pg/ &mu;L). These standards are to be run with each batch of samples prepared to develop the calibration curve used in quantitative analysis.</li>
	<li>For the ISM, aliquot the equivalent volume of 1 &mu;g of each IS (14 total) listed in the reagent table below into a 10 mL volumetric flask, using a calibrated syringe, and dry down under nitrogen. Reconstitute the ISM in 10 mL of ACN and divide into 1 mL aliquots. The final concentration of the ISM should be 100 pg/&mu;L. There is enough ISM to process 1000 samples.</li>
	<li>Store the SM and ISM at -80&deg;C.</li>
</ol>
2. Collection of Media and Cell Lysate
<ol>
	<li>For each 10 cm plate of adherent cells (~ 1 X 107), collect 3 mL of media in a 10 mL glass centrifuge tube. Next, wash the cells 2 times with phosphate buffered saline (PBS). Add 1 mL of PBS to the plate and scrape the cells off the plate and pipet into a second 10 mL glass centrifuge tube. At this point cells and media may be stored if the procedure will not be continued at this time. Store samples at -80&deg;C for long term storage or at -20&deg;C for less than one week.</li>
</ol>
3. Extraction of Lipids from Media
<ol>
	<li>Free bioactive lipids are found in the cell media and these are easily extracted with a one-step l-l extraction procedure. To the collected media (2.1), add 10 &mu;L of ISM. At this time, 8 additional 10 mL glass centrifuge tubes should be labeled #0-7 for the calibration standards. Add 3 mL of PBS to each of these tubes and then add 10 &mu;L of the appropriate SM to tubes 1-7 (1-7 from 1.1).</li>
	<li>Allow the ISM to equilibrate in each calibration standard and sample for 10 minutes and then add 5 mL of diethyl ether. The samples and calibration standards are then placed in the shaker on low speed for 30 minutes at room temperature.</li>
	<li>After shaking, all calibration standards and samples are centrifuged at 1935 x g for 10 minutes. Next, using a Pasteur pipet, transfer the upper phase (organic) into a new 10 mL glass centrifuge tube and dry under nitrogen.</li>
</ol>
4. Extraction of Lipids from Cell Lysate
<ol>
	<li>Spin down the cell samples in the glass centrifuge tubes at 1935 X g for 5 minutes. Dispose of the supernatant; add 2 mL of PBS to each tube, and vortex briefly. Remove 25 &mu;L of sample for protein quantification. Total protein will be used for cell lysate normalization.</li>
	<li>Next, transfer half of the lysate/PBS mixture into a separate 10 mL glass centrifuge tube. Add an additional 2 mL of PBS along with 10 &mu;L of ISM to each sample. Follow the l-l extraction process described for media in 3.2 and 3.3. This aliquot will be used to measure free lipids in the cells and avoid alkaline degradation of lipids, such as the prostaglandins. This aliquot will not undergo steps 4.3 and 4.4 described below.</li>
	<li>The first step of the esterified lipid l-l extraction process requires the addition of 5 mL of CHCl3/MeOH (2:1) to each of the samples. Samples are set in a shaker on low speed for 30 minutes. After shaking, the samples are centrifuged at 1935 x g for 10 minutes. Next, using a Pasteur pipet, transfer the bottom (organic) layer of the l-l extraction into a new 10 mL glass centrifuge tube and dry the sample under nitrogen.</li>
	<li>Esterified lipids must be saponified before quantification. Once the samples are dry, add 0.5 mL of 0.4N KOH in 80% MeOH to each tube. The samples are then incubated at 60&deg;C for 1 h. After the incubation, add 2 mL of PBS. Adjust the pH to 6 with concentrated HCl acid (~30 &mu;L). Add 10 &mu;L of ISM to each sample. The rest of the procedure is the same as lipid extraction from media and can be followed in 3.2 and 3.3.</li>
	<li>If these extractions are run simultaneously with the media samples the same calibration standards can be used to develop a calibration curve; however, if these samples are run at another time, calibration standards must also be processed as described in 3.1-3.3.</li>
</ol>
5. Pentafluorylbenzylbromide (PFB) Derivatization
<ol>
	<li>To the dried down samples and calibration standards, add 100 &mu;L of anhydrous dichloromethane (CH2Cl2) followed by 100 &mu;L of 20% (v/v) N,N-diisopropylethylamine in anhydrous CH2Cl2, and finally 100 &mu;L of 20% (v/v) PFB in anhydrous CH2Cl2.</li>
	<li>After the addition of derivatization reagents, the samples are vortexed, incubated at 60&deg;C for 1 h and dried under nitrogen. The samples and calibration standards may be stored at -80&deg;C if they are not analyzed immediately or at -20&deg;C for up to one week.</li>
</ol>
6. Stable Isotope Dilution Chiral Liquid Chromatography Electron Capture Atmospheric Pressure Chemical Ionization Mass Spectrometry (SID-LC-ECAPCI-MS) Analysis
<ol>
	<li>The dried down samples and calibration standards are reconstituted in 100 &mu;L of hexane/ethanol (97:3). The entire sample can then be transferred to the HPLC vial with insert. Place the samples in the chilled autosampler (4&deg;C) and create a sample list for analysis.</li>
	<li>Next, set up the HPLC and MS methods using the parameters described in Table 1 and Table 2 below. Table 3 contains specific compound information, including the MRM transition and collision energy (CE) for each lipid as well as the estimated retention time from the Waters 2695/Thermo Fisher TSQ Quantum Ultra analysis. The CE is determined by tuning each individual lipid during method development.</li>
	<li>A Chiralpak AD-H column (4.6 X 250 mm, 5 &mu;) heated to 30&deg;C is used for the normal phase separation. The starting mobile phase is 98% hexane and 2% methanol/isopropanol (1:1). After the HPLC separation, methanol is added post-column (0.75 mL/min) to avoid deposition on the Corona needle that would cause a decrease in sensitivity. The MS is set in negative ion mode using the APCI source. If the source needs to be changed from ESI to APCI, be sure to insert a Corona needle.</li>
	<li>The first sample that should be run is always a hexane blank. This will equilibrate the column and also reveal if there is any contamination on the column that may interfere with the calibration standard and sample analysis.</li>
	<li>The next samples run should be the calibration standard samples in order of 0-7 (increasing SM concentration). These standards will be used to create a calibration curve containing the ratio of standard area/IS area on the y-axis and standard concentration or mass on the x-axis. From the y = mx + b equation derived from this plot, the ratio of sample area/IS area (y) can be used to determine sample (unknown) amount or concentration (x).</li>
	<li>The amount of bioactive lipids found in the media can be normalized to the total volume of media and the amount of lipids found in the cell lysate can be normalized to either total number of cells/plate or the amount of total protein/plate. Measures should be taken during 4.1 of the protocol to ensure that cells are counted or that protein is quantified.</li>
</ol>
7. Representative Results:
The chromatograms in Figure 1 are representative of a targeted lipidomics profile. The first panel (Figure 1A) shows 13- and 9-HODE along with the corresponding 13- and 9-oxoODE, derived from linoleic acid. In addition, 15-oxoETE, the oxidized metabolite of 15-HETE is also shown in the second panel. The bottom two panels show 20-HETE and the 20-HETE-IS, [2H6]-20-HETE. Figure 1B shows the separation and unique transitions determined for the HETE stereoisomers that result from enzymatic oxidation of AA or by ROS. Figure 1C is a representation of PGs, isoPs, and LTB4. Some of the standards do not have a deuterated IS commercially available; therefore, an IS that is similar in retention time is used for quantification. For instance, [2H8]-12(S)-HETE is used to quantify 11-HETE and 8-HETE species in addition to 12(R)-HETE and 12(S)-HETE.
From the representative chromatograms, it is evident that there is a small shift in the retention of the deuterated IS compared to the unlabeled standard. This shift to a later retention time is a result of the deuterium interaction with the normal phase column. Deuterium is more polar than hydrogen; therefore, there is a stronger interaction with the normal phase of the column. If this separation were taking place using a reversed phase column, the shift would be to an earlier retention time as a more polar moiety would have a weaker interaction with the reversed phase.
<img src="/files/ftp_upload/3399/3399fig1.jpg" alt="Figure 1" /> 
Figure 1. This figure is a representative example of a targeted lipidomics analysis. Figure 1A shows the total ion chromatograms (TIC) for several LA and AA oxidized metabolites including 13-oxoODE and 9-oxoODE in the first panel, 15-oxoETE in the second panel, 13-HODE and 9-HODE (panels 3 &amp; 5) and 20-HETE (panel 7). The HODEs and 20-HETE have a corresponding deuterated IS in the panel below (4, 6, &amp; 8). The TICs in Figure 2B show the separation and unique transitions of the HETE stereoisomers derived from AA. Figure 2C is a representation of PGs, isoPs, and LTs. The collision energy (CE), MRM transitions, and retention time (RT, min) were optimized using a Waters 2695 LC coupled to a Thermo Fisher TSQ Quantum Ultra and are listed in Table 3 for each compound.
<table border="1">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>% A</td>
			<td>% B</td>
		</tr>
		<tr>
			<td>0</td>
			<td>98</td>
			<td>2</td>
		</tr>
		<tr>
			<td>3</td>
			<td>98</td>
			<td>2</td>
		</tr>
		<tr>
			<td>11</td>
			<td>96.4</td>
			<td>3.6</td>
		</tr>
		<tr>
			<td>15</td>
			<td>92</td>
			<td>8</td>
		</tr>
		<tr>
			<td>27</td>
			<td>92</td>
			<td>8</td>
		</tr>
		<tr>
			<td>30</td>
			<td>50</td>
			<td>50</td>
		</tr>
		<tr>
			<td>38</td>
			<td>50</td>
			<td>50</td>
		</tr>
		<tr>
			<td>39</td>
			<td>98</td>
			<td>2</td>
		</tr>
		<tr>
			<td>45</td>
			<td>98</td>
			<td>2</td>
		</tr>
	</tbody>
</table>
Table 1. HPLC gradient conditions for the chiral normal phase lipidomics separation. Solvent A is 100% hexanes and Solvent B is methanol/isopropanol (1:1).
<table border="1">
	<tbody>
		<tr>
			<td>Mass Spectrometry Parameter</td>
			<td>Setting</td>
		</tr>
		<tr>
			<td>Source</td>
			<td>APCI</td>
		</tr>
		<tr>
			<td>Mode</td>
			<td>Negative Ion</td>
		</tr>
		<tr>
			<td>Method Length</td>
			<td>40 min</td>
		</tr>
		<tr>
			<td>MRM Scan Width</td>
			<td>0.002 m/z</td>
		</tr>
		<tr>
			<td>MRM Scan Time</td>
			<td>0.15 sec</td>
		</tr>
		<tr>
			<td>MRM Peak Width</td>
			<td>0.70 FWHM</td>
		</tr>
		<tr>
			<td>Tube Lens</td>
			<td>164 V</td>
		</tr>
		<tr>
			<td>Source CID</td>
			<td>10 eV</td>
		</tr>
		<tr>
			<td>Q2 Collision Gas</td>
			<td>1.5 units</td>
		</tr>
		<tr>
			<td>Vaporizer Temperature</td>
			<td>450&deg;C</td>
		</tr>
		<tr>
			<td>Capillary Temperature</td>
			<td>250&deg;C</td>
		</tr>
		<tr>
			<td>Discharge Current</td>
			<td>0 &mu;A</td>
		</tr>
		<tr>
			<td>Sheath Gas Pressure</td>
			<td>25 units</td>
		</tr>
		<tr>
			<td>Ion Sweep Pressure</td>
			<td>3 units</td>
		</tr>
		<tr>
			<td>Auxiliary Gas Pressure</td>
			<td>5 units</td>
		</tr>
	</tbody>
</table>
Table 2. Listed above are the optimized mass spectrometry parameters for the measurement of bioactive lipids. These parameters are taken from a Thermo Fisher TSQ Quantum Ultra.
<table border="1">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Transition</td>
			<td>CE (eV)</td>
			<td>RT (min)</td>
		</tr>
		<tr>
			<td>13-oxoODE</td>
			<td>293.03 &rarr; 113.10</td>
			<td>21</td>
			<td>9.9</td>
		</tr>
		<tr>
			<td>9-oxoODE</td>
			<td>293.03 &rarr; 113.10</td>
			<td>21</td>
			<td>17.9</td>
		</tr>
		<tr>
			<td>15-oxoETE</td>
			<td>317.05 &rarr; 113.18</td>
			<td>18</td>
			<td>8.1</td>
		</tr>
		<tr>
			<td>13(R)-HODE</td>
			<td>295.03 &rarr; 195.12</td>
			<td>18</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>13(S)-HODE</td>
			<td>295.03 &rarr; 195.12</td>
			<td>18</td>
			<td>14.8</td>
		</tr>
		<tr>
			<td>[2H4]-13(S)-HODE</td>
			<td>299.03 &rarr; 198.12</td>
			<td>18</td>
			<td>14.9</td>
		</tr>
		<tr>
			<td>9(R)-HODE</td>
			<td>295.03 &rarr; 171.10</td>
			<td>18</td>
			<td>13.7</td>
		</tr>
		<tr>
			<td>9(S)-HODE</td>
			<td>295.03 &rarr; 171.10</td>
			<td>18</td>
			<td>21.2</td>
		</tr>
		<tr>
			<td>[2H4]-9(S)-HODE</td>
			<td>299.03 &rarr; 172.12</td>
			<td>18</td>
			<td>21.2</td>
		</tr>
		<tr>
			<td>20-HETE</td>
			<td>319.03 &rarr; 289.10</td>
			<td>18</td>
			<td>12.4</td>
		</tr>
		<tr>
			<td>[2H6]-20-HETE</td>
			<td>325.03 &rarr; 295.10</td>
			<td>18</td>
			<td>12.4</td>
		</tr>
		<tr>
			<td>5(R)-HETE</td>
			<td>319.04 &rarr; 115.04</td>
			<td>16</td>
			<td>15.7</td>
		</tr>
		<tr>
			<td>5(S)-HETE</td>
			<td>319.04 &rarr; 115.04</td>
			<td>16</td>
			<td>16.2</td>
		</tr>
		<tr>
			<td>[2H8]-5(S)-HETE</td>
			<td>327.04 &rarr; 116.04</td>
			<td>16</td>
			<td>16.4</td>
		</tr>
		<tr>
			<td>12(R)-HETE</td>
			<td>319.04 &rarr; 179.11</td>
			<td>14</td>
			<td>11.7</td>
		</tr>
		<tr>
			<td>12(S)-HETE</td>
			<td>319.04 &rarr; 179.11</td>
			<td>14</td>
			<td>12.3</td>
		</tr>
		<tr>
			<td>[2H8]-12(S)-HETE</td>
			<td>327.13 &rarr; 184.11</td>
			<td>14</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>15(R)-HETE</td>
			<td>319.04 &rarr; 219.12</td>
			<td>13</td>
			<td>12.4</td>
		</tr>
		<tr>
			<td>15(S)-HETE</td>
			<td>319.04 &rarr; 219.12</td>
			<td>13</td>
			<td>15.9</td>
		</tr>
		<tr>
			<td>[2H8]-15(S)-HETE</td>
			<td>327.03 &rarr; 226.12</td>
			<td>13</td>
			<td>16.1</td>
		</tr>
		<tr>
			<td>11(R)-HETE</td>
			<td>319.04 &rarr; 167.11</td>
			<td>16</td>
			<td>11.1</td>
		</tr>
		<tr>
			<td>11(S)-HETE</td>
			<td>319.04 &rarr; 167.11</td>
			<td>16</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>8(R)-HETE</td>
			<td>319.04 &rarr; 155.09</td>
			<td>16</td>
			<td>11.7</td>
		</tr>
		<tr>
			<td>8(S)-HETE</td>
			<td>319.04 &rarr; 155.09</td>
			<td>16</td>
			<td>12.6</td>
		</tr>
		<tr>
			<td>LTB4</td>
			<td>335.03 &rarr; 195.10</td>
			<td>18</td>
			<td>32.0</td>
		</tr>
		<tr>
			<td>[2H4]-LTB4</td>
			<td>339.03 &rarr; 197.10</td>
			<td>18</td>
			<td>32.0</td>
		</tr>
		<tr>
			<td>PGE2</td>
			<td>351.03 &rarr; 271.15</td>
			<td>18</td>
			<td>31.2</td>
		</tr>
		<tr>
			<td>[2H4]-PGE2</td>
			<td>355.03 &rarr; 275.15</td>
			<td>18</td>
			<td>31.2</td>
		</tr>
		<tr>
			<td>PGD2</td>
			<td>351.03 &rarr; 271.15</td>
			<td>25</td>
			<td>32.5</td>
		</tr>
		<tr>
			<td>[2H4]-PGD2</td>
			<td>355.03 &rarr; 275.15</td>
			<td>25</td>
			<td>32.5</td>
		</tr>
		<tr>
			<td>8-iso-PGE2</td>
			<td>351.03 &rarr; 271.15</td>
			<td>18</td>
			<td>37.4</td>
		</tr>
		<tr>
			<td>11&beta;-PGF2</td>
			<td>353.03 &rarr; 193.11</td>
			<td>25</td>
			<td>27.6</td>
		</tr>
		<tr>
			<td>[2H4]-11&beta;-PGF2</td>
			<td>357.03 &rarr; 197.11</td>
			<td>25</td>
			<td>27.6</td>
		</tr>
		<tr>
			<td>PGF2&alpha;</td>
			<td>353.03 &rarr; 193.11</td>
			<td>25</td>
			<td>33.1</td>
		</tr>
		<tr>
			<td>[2H4]-PGF2&alpha;</td>
			<td>357.03 &rarr; 197.11</td>
			<td>25</td>
			<td>33.1</td>
		</tr>
		<tr>
			<td>8-iso-PGF2&alpha;</td>
			<td>353.03 &rarr; 193.11</td>
			<td>25</td>
			<td>34.4</td>
		</tr>
		<tr>
			<td>[2H4]-8-iso-PGF2&alpha;</td>
			<td>357.03 &rarr; 197.11</td>
			<td>25</td>
			<td>34.4</td>
		</tr>
	</tbody>
</table>
Table 3. This table lists the compounds detected in the total ion chromatograms of Figure 1A-C. The second column provides the transition used for MRM/MS, the third column lists the collision energy (CE, eV) and the last column lists the retention time (RT, min). Parameters were optimized using a Waters 2695 separation module coupled to a Thermo Fisher TSQ Quantum Ultra.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable-isotope+dilution+LC-MS+for+quantitative+biomarker+analysis.">Stable-isotope dilution LC-MS for quantitative biomarker analysis.</a> Bioanalysis. 2, 311-341 (2010).</li><li>Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron-capture+negative-ion+chemical+ionization+mass-spectrometry+of+lipid+mediators.">Electron-capture negative-ion chemical ionization mass-spectrometry of lipid mediators.</a> Methods in Enzymology. 187, 13-23 (1990).</li><li>Singh, G., Gutierrez, A., Xu, K., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography/electron+capture+atmospheric+pressure+chemical+ionization/mass+spectrometry:+analysis+of+pentafluorobenzyl+derivatives+of+biomolecules+and+drugs+in+the+attomole+range.">Liquid chromatography/electron capture atmospheric pressure chemical ionization/mass spectrometry: analysis of pentafluorobenzyl derivatives of biomolecules and drugs in the attomole range.</a> Analytical Chemistry. 72, 3007-3013 (2000).</li><li>Blair, I. A., Capriol, R. M., Gross, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Mass Spectrometry. , 283-307 (2005).</li><li>Lee, S. H., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+chiral+lipidomics+analysis+by+liquid+chromatography+electron+capture+atmospheric+pressure+chemical+ionization+mass+spectrometry+(LC-ECAPCI/MS).">Targeted chiral lipidomics analysis by liquid chromatography electron capture atmospheric pressure chemical ionization mass spectrometry (LC-ECAPCI/MS).</a> Methods in Enzymology. 433, 159-174 (2007).</li></ol>

No conflicts of interest declared.

The standards and internal standards used in this protocol provide a representation of a targeted lipidomics method. A Waters 2695 separation module and Thermo Fisher TSQ Quantum Ultra were used for the LC-MS analysis and the optimal parameter settings can be found in Tables 1 and 2. Additionally, this extraction protocol was designed for adherent cells, but can be modified for other cell types as well as other biological matrices including urine, blood, and tissue. Many lipid standards ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic-6,8,9,11,12,14,15-d7 acid</td>
			<td>Cayman Chemical</td>
			<td>334250</td>
			<td>[2H7]-5-oxoETE Internal Standard</td>
		</tr>
		<tr>
			<td>5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid</td>
			<td>Cayman Chemical</td>
			<td>334230</td>
			<td>[2H8]-5(S)-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid</td>
			<td>Cayman Chemical</td>
			<td>334570</td>
			<td>[2H8]-12(S)-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid</td>
			<td>Cayman Chemical</td>
			<td>334720</td>
			<td>[2H8]-15(S)-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic-16,16,17,17,18,18-d6 acid</td>
			<td>Cayman Chemical</td>
			<td>390030</td>
			<td>[2H6]-20-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>9S-hydroxy-10E,12Z-octadecadienoic-9,10,12,13-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>338410</td>
			<td>[2H4]-9(S)-HODE Internal Standard</td>
		</tr>
		<tr>
			<td>13S-hydroxy-9Z,11E-octadecadienoic-9,10,12,13-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>338610</td>
			<td>[2H4]-13(S)-HODE Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>316010</td>
			<td>[2H4]-PGF2&alpha; Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-(8&beta;)-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>316350</td>
			<td>[2H4]-8-iso-PGF2a Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&beta;.,15S-trihydroxy-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>10008989</td>
			<td>[2H4]-11&beta;-PGF2 Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>312010</td>
			<td>[2H4]-PGD2 Internal Standard</td>
		</tr>
		<tr>
			<td>9-oxo-11&alpha;,15S-dihydroxy-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>314010</td>
			<td>[2H4]-PGE2 Internal Standard</td>
		</tr>
		<tr>
			<td>5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic-6,7,14,15-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>320110</td>
			<td>[2H4]-LTB4 Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11,15S-trihydroxy-thromba-5Z,13E-dien-1-oic-3,3,4,4-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>319030</td>
			<td>[2H4]-TxB2 Internal Standard</td>
		</tr>
		<tr>
			<td>5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34250</td>
			<td>5-oxoETE Standard</td>
		</tr>
		<tr>
			<td>12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34580</td>
			<td>12-oxoETE Standard</td>
		</tr>
		<tr>
			<td>15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34730</td>
			<td>15-oxoETE Standard</td>
		</tr>
		<tr>
			<td>5R-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34225</td>
			<td>5(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34230</td>
			<td>5(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>8R-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34350</td>
			<td>8(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>8S-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34360</td>
			<td>8(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34505</td>
			<td>11(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>11S-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34510</td>
			<td>11(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34560</td>
			<td>12(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34570</td>
			<td>12(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>15R-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34710</td>
			<td>15(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34720</td>
			<td>15(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>90030</td>
			<td>20-HETE Standard</td>
		</tr>
		<tr>
			<td>9R-hydroxy-10E,12Z-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38405</td>
			<td>9(R)-HODE Standard</td>
		</tr>
		<tr>
			<td>9S-hydroxy-10E,12Z-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38410</td>
			<td>9(S)-HODE Standard</td>
		</tr>
		<tr>
			<td>13R-hydroxy-9Z,11E-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38605</td>
			<td>13(R)-HODE Standard</td>
		</tr>
		<tr>
			<td>13S-hydroxy-9Z,11E-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38610</td>
			<td>13(S)-HODE Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>16010</td>
			<td>PGF2&alpha; Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-(8&beta;)-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>16350</td>
			<td>8-iso-PGF2&alpha; Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&beta;,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>16520</td>
			<td>11&beta;-PGF2 Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>12010</td>
			<td>PGD2 Standard</td>
		</tr>
		<tr>
			<td>9-oxo-11&alpha;,15S-dihydroxy-(8&beta;)-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>14350</td>
			<td>8-iso-PGE2 Standard</td>
		</tr>
		<tr>
			<td>9-oxo-11&alpha;,15S-dihydroxy-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>14010</td>
			<td>PGE2 Standard</td>
		</tr>
		<tr>
			<td>5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>20110</td>
			<td>LTB4 Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11,15S-trihydroxythromba-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>19030</td>
			<td>TxB2 Standard</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Diethyl Ether</td>
			<td>Sigma</td>
			<td>346136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Acros</td>
			<td>61030-1000</td>
			<td>anhydrous</td>
		</tr>
		<tr>
			<td>N,N-diisopropylethyl amine</td>
			<td>Sigma</td>
			<td>387649</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pentafluorylbenzyl bromide</td>
			<td>Sigma</td>
			<td>101052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma</td>
			<td>320331</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fluka</td>
			<td>00650</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>A996-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A454-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>366927</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher</td>
			<td>H303-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher</td>
			<td>A464-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Fisher</td>
			<td>W7-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pasteur Pipets</td>
			<td>Fisher</td>
			<td>13-678-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 mL Glass Centrifuge Tubes</td>
			<td>Kimble</td>
			<td>73785-10</td>
			<td>Screw cap</td>
		</tr>
		<tr>
			<td>Phenolic Screw Caps</td>
			<td>Kimble</td>
			<td>73802-13415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chiralcel ADH Column</td>
			<td>Chiral Technologies</td>
			<td>19325</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC vials</td>
			<td>Waters</td>
			<td>60000751CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC inserts</td>
			<td>Waters</td>
			<td>WAT094171</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis at JoVE.com

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

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 ...

cellular-lipid-extraction-for-targeted-stable-isotope-dilution-liquid

Research

JoVE Journal

Bioengineering

15.1K Views.  University of Pennsylvania. 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).

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

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

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 ...

Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal resolution from shallow and deep brain regions. Their use has been hindered, however, by mechanical and structural mismatches between the probes and brain tissue that commonly lead to micromotion and gliosis with resulting signal instability in chronic recording experiments. In contrast, following the implantation of ultraflexible mesh electronics via syringe injection, the mesh probes form a seamless, gliosis-free interface with the surrounding brain tissue that enables stable tracking of individual neurons on at least a year timescale. This protocol details the key steps in a typical mouse neural recording experiment using syringe-injectable mesh electronics, including the fabrication of mesh electronics in a standard photolithography-based process possible at many universities, loading mesh electronics into standard capillary needles, stereotaxic injection in vivo, connection of the mesh input/output to standard instrumentation interfaces, restrained or freely moving recording sessions, and histological sectioning of brain tissue containing mesh electronics. Representative neural recordings and histology data are presented. Investigators familiar with this protocol will have the knowledge necessary to incorporate mesh electronics into their own experiments and take advantage of the unique opportunities afforded by long-term stable neural interfacing, such as studies of aging processes, brain development, and the pathogenesis of brain disease.

Mesh electronics probes seamlessly integrate and provide stable, long-term, single-neuron level recording within the brain. This protocol uses mesh electronics for in vivo experiments, involving the fabrication of mesh electronics, loading into needles, stereotaxic injection, input/output interfacing, recording experiments, and histology of tissue containing mesh probes.

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

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.

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 ...