We would like to thank Dr. Evan Newell and Dr. Sean Bendall for feedback. We would also like to thank DVS Sciences and Dr. Sean Bendall for a sample of the Multi-lanthanide-containing polystyrene beads. We are grateful for funding from NIH grant 2 U19 AI057229.

All cell samples for the CyTOF must be fixed and permeabilized. This enables greater entry of the iridium-containing DNA intercalator, and also prevents cell lysis during the MilliQ water wash and resuspension steps immediately before injecting into the mass cytometer.
1. Start-up of the Mass Cytometer
<ol>
 <li>Open the CyTOF software program. Select Instrument Setup. On the Card Cage tab, go to the lower right corner and click Heater "ON" button. Turn on the heater at least 15 min prior to desired time to allow sufficient time for the spray chamber mantle to reach 200 &deg;C.</li>
 <li>Replace the white Norm-Ject 3 ml syringe in the syringe pump with a fresh syringe filled with MilliQ water.</li>
 <li>Cleaning nebulizer. Backflush the nebulizer by using the syringe and tubing to pull 5% citranox 2-3 times through both the smaller liquid introduction port and the larger gas introduction port. Repeat backflushing with MilliQ water to remove residual citranox.</li>
 <li>Check to confirm that the lights on the front panel of the CyTOF are lit as in Figure 1.</li>
 </ol>
 <img alt="Figure 1" src="/files/ftp_upload/4398/4398fig1.jpg" /> 
 Figure 1. CyTOF panel lights prior to startup.
 <ol start="5">
 <li>Open argon gas tank valve. This should cause the "ARGON" light on the front to turn on.</li>
 <li>Attach the nebulizer to make-up gas inlet. Unscrew the make-up gas tubing connector and remove the black rubber o-ring and the white conical plastic piece. Note the wider "knuckle" on the gas introduction port stem of the nebulizer. Place the connector on the gas introduction port. Place the black o-ring over the end of the gas port, and force the o-ring past the wider part of the port stem. Place the white conical plastic piece on top with the narrow end pointed out, and screw onto the make-up gas tubing.</li>
 <li>Attach the nebulizer to the liquid introduction tubing. Carefully insert the small tubing into the opening at the end of the nebulizer. There will be two stops, similar in feel to the stops on a micropipettor. First, press until you meet light resistance; the end of the small tubing should be at the end of the straight section of glass. Second, press harder to force the end of the straight section of glass further into the fitting, and tighten the screw fitting to seal. Insert the end of the nebulizer into the white opening in the heating mantle.</li>
 <li>On the RFG Controller tab of the Instrument Setup window, press "Start Plasma". Since the nebulizer is already inserted, press "OK". The software will turn on the chiller, start the flow of gas, and ignite a plasma. The detector voltage will ramp up. There will be a pop-up window saying "Plasma start-up sequence has finished successfully" when finished; press "OK". New lights should be on in the front panel (Figure 2).</li>
 </ol>
<img alt="Figure 2" src="/files/ftp_upload/4398/4398fig2.jpg" /> 
 Figure 2. CyTOF panel lights after plasma ignited and start-up successfully completed.
 <ol start="9">
 <li>Turning on syringe pump. In the upper right corner, press the green "play" button to start the syringe pump and begin flowing liquid through the tubing and spraying out of the nebulizer. The default syringe settings are for 3 ml of water. In order for sample to be flowing through the sample loop, the syringe pump must be periodically changed when it runs out of water. At the top of the screen, there is a counter indicating how much liquid has flowed out of the syringe pump. The syringe pump will stop when this counter reaches "3.000" or the plunger hits the end of the syringe. When refilling the syringe, you must hit "stop" rather than the blue "pause" button to reset the counter.</li>
</ol>
2. Daily Calibration of the Mass Cytometer
<ol>
 <li>The mass cytometer will need approximately 20 min to achieve a hot, stable plasma before calibration. The purpose of the tuning steps is to maximize the desired signal of Tm169 or Tb159 while keeping "Gd155" (oxide; La139+O16) signal under 3% of the higher value. Click the Acquisition Settings button to open the Data Acquisition Settings window. In the Analytes tab, click the periodic table to open the isotope selection panel. Select the isotopes in the DVS Sciences tuning solution listed in the manual, then click "save".</li>
 <li>Click the Isotopes Per Reading button to open the Mass(es) Per Reading window. Select "Pulses Count", and rescale the y-axis by entering a new number and pressing "enter".</li>
 <li>Fill a green 1 ml Norm-Ject syringe with tuning solution. Turn the sample port selector to "LOAD", inject 450 &mu;l of tuning solution, then turn the selector to "INJECT". In the Mass Calibration tab of the Data Acquisition Settings window, press "Run" to monitor which masses are flowing by. The left side of the window is lower mass, while the right side is higher mass. Note: there will already be signals in the TOF 9400-9800 region, due to xenon isotopes in the argon gas (Figure 3). Wait until tuning solution isotopes start appearing as streaks in this display (Figure 4).</li>
 </ol>
 <img alt="Figure 3" src="/files/ftp_upload/4398/4398fig3.jpg" /> 
 Figure 3. Mass calibration tab window, showing background levels for various isotopes after wash solution and water rinse. The region around TOF 9400-9800 corresponds to xenon isotopes in the argon gas, and should always be present. <a href="https://www.jove.com/files/ftp_upload/4398/4398fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4398/4398fig4.jpg" /> 
 Figure 4. Mass calibration tab window, showing vertical streaks for various isotopes in DVS tuning solution. <a href="https://www.jove.com/files/ftp_upload/4398/4398fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
 <ol start="4">
 <li>Go back to the Mass(es) Per Reading window and press the green "Play" button in the upper left corner. Each isotope selected in Step 2.1 is represented by a different colored line. The default x-axis is "Time", in sec. Monitor the levels of the different isotopes until they are steady.</li>
 <li>Tuning of the current. On the Parameter tab of the Data Acquisition Settings window, select "Current" from the drop-down menu. Let start=0, finish=10, step value=0.5, settling time=200, and press "save". Click back on the Mass(es) Per Reading window and hit play. The x-axis is now "Current". Note where the signals of the isotopes peak (Figure 5), and record. Under the DAC Channels Setup tab of the Instrument Setup window, scroll down to "Current" and enter this new value. Press "Set actual current value", then "save".</li>
 </ol>
 <img alt="Figure 5" src="/files/ftp_upload/4398/4398fig5.jpg" /> 
 Figure 5. Current tuning profile.
 <ol start="6">
 <li>Tuning of the make-up gas. Change drop-down menu to "Make-up gas." Let start=0.55, end=0.95, step value=0.05, settling time=200. Press save. Return to the Mass(es) Per Reading window and hit play. The x-axis is now "Make-up gas" flow rate. Record where the signals of the isotopes peak. Observe the rapid increase of the "Gd155" signal near the end (Figure 6). Return to the DAC Channels Setup tab, scroll down to "Make-up gas", and enter this new value. Press "Set actual current value", then "save".</li>
 </ol>
<img alt="Figure 6" src="/files/ftp_upload/4398/4398fig6.jpg" /> 
 Figure 6. Make-up gas tuning profile.
 <ol start="7">
 <li>Recording ratio of desired isotope vs oxide. Make a second 450 &mu;l injection of tuning solution. On the Parameter tab, select "Time" from the drop-down menu, let start=0, end=10, step value=1, settling time=200. Press "save." Hit "play" in Mass(es) Per Reading window. After the lines have stabilized (Figure 7), use the cursor to read the value in the upper left boxes for Tm169 or Tb159 (higher value) and also the "Gd155" value (&lt;3% of the higher value). These values can also be saved in a tuning file for future reference.</li>
 </ol>
 <img alt="Figure 7" src="/files/ftp_upload/4398/4398fig7.jpg" /> 
 Figure 7. Measurement of tuning solution signal intensities after optimizing Current and Make-up gas.
 <ol start="8">
 <li>Cleaning of sample loop. Inject 3 ml of MilliQ water through the sample loop, followed by 500 &mu;l of DVS Wash Solution. Monitor the progress of the cleaning by pressing "run" in the Mass Calibration tab. Wash until the intensity of the streaks (Figure 4) begins to decrease, then follow by 3 ml of MilliQ water and monitor until down to background levels (Figure 3).</li>
</ol>
3. Running Samples
<ol>
 <li>Open the Acquisition window. Default settings: Do Analysis = On-The-Fly, Signal to Analyze = Dual, Dual Count Calibration = Data. Noise Reduction is selected. Any of these may be changed if desired. Enter the file name. All files MUST be saved to the E:// drive.</li>
 <li>Setting acquisition delays. There will be a slight delay in registering cell events of about 30 sec due to the length of the tubing between the sample loop and the nebulizer. Therefore, the sum total of "Acquisition delay" and "Detector stability delay" should be at least 30 sec. Detector stability delay should be at least 20 sec.</li>
 <li>Setting data collection time. "Acquisition time" is the number of seconds you want to run your sample. Filling the entire 450 &mu;l sample loop would require 600 sec to complete. It is suggested that 50-100 sec be added to the end of the run time to minimize sample carry-over.</li>
 <li>Resuspension of sample. To minimize build-up of salts in the machine, it is suggested that at least two washes in MilliQ water be done at the end of the cell staining, followed by a final resuspension in MilliQ water. The volume of MilliQ water used to resuspend the cell pellet will vary depending on the number of cells you started with and the cell recovery after the entire staining procedure. A good starting place is 106 (starting) cells/ml. After resuspension, filter through a cell strainer to remove aggregates.</li>
 <li>Starting sample run. Enter a new file name for the current sample. Switch the sample loop valve to "LOAD", inject the sample, switch back to "INJECT", then hit "Run" in the Acquisition window.</li>
 <li>Registering cell events. Cells will be represented in the snapshot window as horizontal collections of marks in each vertical isotope/marker channel (Figure 8). While the mass cytometer can register 1,000 cells/sec, better resolution between cell events is usually obtained at 300-500 cells/sec. This corresponds to an average of ~1 cell/screen refresh. It may take up to 30 sec before the cell event counter starts to climb. This is due to "On-the-fly" data processing; no cell event information is lost.</li>
 </ol>
<img alt="Figure 8" src="/files/ftp_upload/4398/4398fig8.jpg" /> 
 Figure 8. Acquisition window during sample running, showing horizontal rows of spots corresponding to the elements associated with three separate cell events. <a href="https://www.jove.com/files/ftp_upload/4398/4398fig8large.jpg" target="_blank"> Click here to view larger figure</a>.
 <ol start="7">
 <li>Quality of cell events. Notice any background streaking in each isotope channel, as well as the cell event rate. The mass cytometer recognizes a "cell event" as a certain increase in signal above background, so background signal from free metal or unbound antibody can alter the "cell event" count. Additionally, running cells at too high a concentration may cause an increased number of doublet events. Background can be diminished by additional washes or greater cell dilution. Doublets can be minimized by greater cell dilution, at the cost of increased run time.</li>
 <li>Finishing sample data acquisition. When the Acquisition time has been reached, the data collection will finish. There will be a bit of delay before a pop-up window with an "OK" button appears; again, this is due to "On-the-Fly" data processing. Sample acquisition can also be stopped prematurely by hitting the "Stop" button. In this case, all the cell events already registered will be counted and processed into output files.</li>
 <li>Injection of at least 500 &mu;l of MilliQ water between each sample also helps minimize sample carryover.</li>
</ol>
4. Cleaning the Machine After Use
<ol>
 <li>Wash solution. After the final sample is run and the sample loop flushed with water, inject 450 &mu;l of wash solution into the sample loop. Monitor the release of free metal in the Mass Calibration tab of the Data Acquisition Settings window as in Step 2.8 (Figure 9). When the free metal signal begins to decrease, flush with 3 ml of MilliQ water and monitor until background levels are achieved.</li>
</ol>
<img alt="Figure 9" src="/files/ftp_upload/4398/4398fig9.jpg" /> 
 Figure 9. Mass calibration tab window, during post-run cleaning with DVS Wash solution. The vertical streaks in TOF region 9800-11000 are antibody-label isotopes being cleaned out of the machine. The vertical streaks around TOF 11500 correspond to the two Ir intercalator isotopes. <a href="https://www.jove.com/files/ftp_upload/4398/4398fig9large.jpg" target="_blank"> Click here to view larger figure</a>.
5. Shutting Down the Machine
<ol>
 <li>In the RFG Controller tab of the Instrument Setup window, select "Stop Plasma". When the procedure is complete, there will be a pop-up window asking you to remove the nebulizer. Press "OK". In the Card Cage tab, turn off the heater. Close the valve on the argon supply.</li>
 <li>Removal of nebulizer. Remove the nebulizer from the spray chamber by carefully pulling backwards. Unscrew the sample introduction tubing from Step 1.6 and place aside. Unscrew the make-up gas introduction connection to loosen slightly, then pull the nebulizer off. Leaving the fitting screwed together will reduce the chances of losing the black rubber o-ring and white conical plastic piece.</li>
 <li>Cleaning nebulizer. Backflush both ports of the nebulizer with 5% citranox using the syringe and tubing as in Step 1.3. Store, covered, in 5% citranox until next use.</li>
</ol>

<ol>
	<li>Bandura, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+cytometry:+Technique+for+real+time+single+cell+multitarget+immunoassay+based+on+inductively+coupled+plasma+Time-of-Flight+mass+spectrometry.">Mass cytometry: Technique for real time single cell multitarget immunoassay based on inductively coupled plasma Time-of-Flight mass spectrometry.</a> Anal. Chem. 81, 6813-6822 (2009).</li><li>Lou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-based+elemental+tags+for+sensitive+bioassays.">Polymer-based elemental tags for sensitive bioassays.</a> Angew. Chem., Int. Ed. 46, 6111-6114 (2007).</li><li>Majonis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curious+results+with+palladium-+and+platinum-carrying+polymers+in+mass+cytometry+bioassays+and+an+unexpected+application+as+a+dead+cell+stain.">Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain.</a> Biomacromolecules. 12, 3997-4010 (2011).</li><li>Leipold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+mass+cytometry+methods+for+bacterial+discrimination.">Development of mass cytometry methods for bacterial discrimination.</a> Anal. Biochem. 419, 1-8 (2011).</li><li>Bendall, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mass+cytometry+of+differential+immune+and+drug+responses+across+a+human+hematopoietic+continuum.">Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.</a> Science. 332, 687-696 (2011).</li><li>Newell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry+by+Time-of-Flight+shows+combinatorial+cytokine+expression+and+virus-specific+cell+niches+within+a+continuum+of+CD8++T+cell+phenotypes.">Cytometry by Time-of-Flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes.</a> Immunity. 36, 142-152 (2012).</li></ol>

The authors both work at the Human Immune Monitoring Center, a service center at Stanford University which charges user fees solely to recover the cost of assays, including mass cytometry.

For the last few decades, fluorescence flow cytometry has been a workhorse method for analyzing single cells, both in terms of surface expression and in functional assays. However, the issues of spectral overlap of the fluorescent dyes has limited the number of simultaneous markers. While experiments using more than 12 simultaneous markers have been reported, the amount of compensation necessary makes this technically difficult.
Instead of fluorophores, mass cytometry pioneered by DVS Sciences...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>CyTOF mass cytometer</td>
 <td>DVS Sciences</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wash solution</td>
 <td>DVS Sciences</td>
 <td>201071</td>
 <td>0.05% hydrofluoric acid in water</td>
 </tr>
 <tr>
 <td>Tuning solution</td>
 <td>DVS Sciences</td>
 <td>201072</td>
 <td>0.5 ppm La, Cs, Tb, Tm, Ir, trace nitric acid</td>
 </tr>
 <tr>
 <td>Eu-containing polystyrene beads</td>
 <td>DVS Sciences</td>
 <td>201073</td>
 <td>Contains natural-abundance Eu isotopes; beads supplied at approximately 1 million/ml</td>
 </tr>
 <tr>
 <td>Multi-lanthanide-containing (La/Pr/Tb/Tm/Lu)- polystyrene beads</td>
 <td>DVS Sciences</td>
 <td>Not yet commercially-available</td>
 <td>Contains natural-abundance isotopes of listed lanthanides</td>
 </tr>
 <tr>
 <td>Nitric acid (concentrated)</td>
 <td>Fisher Scientific</td>
 <td>A467-500</td>
 <td>Optima trace-metal pure ICP-MS grade</td>
 </tr>
 <tr>
 <td>Polystyrene round-bottom tube with cell-strainer cap-5 ml</td>
 <td>BD</td>
 <td>352235</td>
 <td>used to filter cell samples before injection</td>
 </tr>
 <tr>
 <td>Norm-Ject tuberkulin syringe-1 ml</td>
 <td>Henke Sass Wolf</td>
 <td>4010-200V0</td>
 <td>silicone-free, latex-free</td>
 </tr>
 <tr>
 <td>Norm-Ject syringe-3 ml</td>
 <td>Henke Sass Wolf</td>
 <td>4010.000V0</td>
 <td>silicone-free, latex-free</td>
 </tr>
 <tr>
 <td>MilliQ water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>18 M&Omega; pure water; must not be stored in glass or plastic bottles that have been washed with commercial detergent (due to their high levels of barium present).</td>
 </tr>
 <tr>
 <td>Citranox</td>
 <td>Sigma-Aldrich</td>
 <td>Z273236</td>
 <td>acid detergent</td>
 </tr>
 <tr>
 <td>Argon gas</td>
 <td>Praxair</td>
 <td>AR 5.0UH-T</td>
 <td>99.999% Ultra-high purity</td>
 </tr>
 </tbody>
</table>

mass cytometry: protocol for daily tuning and running cell samples on a cytof mass cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

Following the above protocol should accomplish four things. First, allowing adequate warm-up time for the mass cytometer will produce a hot, stable plasma necessary for optimal signal and minimal oxide formation. Second, adequate washing of the mass cytometer with MilliQ water and DVS Wash solution (Figure 9) will help reduce the levels of metal adsorption to the tubing and other parts of the machine, helping to reduce background during sample acquisition (Figure 8). It will also help re...

Watch this Scientific Journal Video about Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer at JoVE.com

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

mass-cytometry-protocol-for-daily-tuning-running-cell-samples-on

Research

JoVE Journal

Bioengineering

22.7K Views.  Stanford University. Шаги, необходимые для ежедневной настройки и оптимизации производительности цитометр массы CyTOF описаны. Комментарии по оптимальной подготовки проб и расхода обсуждается

Video: Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

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

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

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

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

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

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

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

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

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

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

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

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...