Name: Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer (Video) | JoVE Journal
Uploaded: 2012-11-02T14:00:00.000+00:00
Duration: 10 min 59 s
Description: Learn daily tuning and sample analysis on a CyTOF mass cytometer for optimal cell data acquisition. This protocol demonstrates instrument setup, signal optimization, and proper cleaning procedures.

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5 minutes

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 °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="Mass cytometer control panel, CyTOF setup for cellular analysis, plasma off indicator." src="/files/ftp_upload/4398/4398fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 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="CyTOF mass cytometer control panel with indicators for plasma, cooling, and argon gas status." src="/files/ftp_upload/4398/4398fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
 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 μl 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="Mass spectrometry TOF diagram; data acquisition setup; mass calibration; time-of-flight analysis." src="/files/ftp_upload/4398/4398fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
 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="Time-of-flight mass spectrometry graph; spectral data analysis showing ion separation." src="/files/ftp_upload/4398/4398fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
 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="Mass spectrometry graph, intensity vs mass, showing isotopes (Xe, Cs, La) in pulse count analysis." src="/files/ftp_upload/4398/4398fig5.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
 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="Mass spectrometry pulse count graph for elements, Xe to Ir, showing spectral intensity variation." src="/files/ftp_upload/4398/4398fig6.jpg" data-altupdatedat="1787630408000" data-alt="Figure 6"> 
 Figure 6. Make-up gas tuning profile.
 <ol start="7">
 <li>Recording ratio of desired isotope vs oxide. Make a second 450 μl 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="Mass spectrometry graph shows isotope pulse counts over time for elements like Xe and Gd." src="/files/ftp_upload/4398/4398fig7.jpg" data-altupdatedat="1787630408000" data-alt="Figure 7"> 
 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 μl 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 μl 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="Flow cytometry data analysis, fluorescence intensity graph, cell population quantification system." src="/files/ftp_upload/4398/4398fig8.jpg" data-altupdatedat="1787630408000" data-alt="Figure 8"> 
 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 μl 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 μl 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="Mass spectrometry data acquisition screen, TOF graph, mass calibration results, ion detection." src="/files/ftp_upload/4398/4398fig9.jpg" data-altupdatedat="1787630408000" data-alt="Figure 9"> 
 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/pubmed?term=((Mass+cytometry%3A+Technique+for+real+time+single+cell+multitarget+immunoassay+based+on+inductively+coupled+plasma+Time-of-Flight+mass+spectrometry%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">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/pubmed?term=((Polymer-based+elemental+tags+for+sensitive+bioassays%5BTitle%5D)+AND+%22Angew.+Chem.%2C+Int.+Ed%22%5BJournal%5D)">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/pubmed?term=((Curious+results+with+palladium-+and+platinum-carrying+polymers+in+mass+cytometry+bioassays+and+an+unexpected+application+as+a+dead+cell+stain%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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/pubmed?term=((Development+of+mass+cytometry+methods+for+bacterial+discrimination%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">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/pubmed?term=((Single-cell+mass+cytometry+of+differential+immune+and+drug+responses+across+a+human+hematopoietic+continuum%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Cytometry+by+Time-of-Flight+shows+combinatorial+cytokine+expression+and+virus-specific+cell+niches+within+a+continuum+of+CD8%2B+T+cell+phenotypes%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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><tbody><tr><td>CyTOF mass cytometer</td><td>DVS Sciences</td><td></td><td></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></td><td></td><td>18 M&amp;#937; 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

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...

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 ...

The overhaul goal of this procedure is to demonstrate the proper daily tuning of and sample analysis on a CyTOF instrument. First, the machine is assembled and turned on. Next, the instrument settings are optimized for tuning to the maximum signal.The machine is then ready to run the samples. In the final step, the machine is cleaned and shut down. Ultimately, optimal cell data can be acquired according to specific time requirements.Visual demonstration of this technique is critical. The daily setup, tuning and cleaning procedures are difficult to learn from the manual. As there are multiple simultaneous software steps involved.You need to gain experience to acquire a feel for the appropriate background levels and signal intensities At least 15 minutes prior to beginning an experiment. Open the CyTOF software program and select instrument setup. Then in the card cage tab, click the heater on button to allow the spray chamber mantle to reach 200 degrees Celsius.Replace the syringe in the syringe pump with a fresh syringe filled with milli cube water. Then clean the nebulizer by back flushing with 5%Citron twice through reach port. To remove any residual Citron, repeat the back flush with Milli Q water.Next, open the argon gas tank valve resulting in the Argonne light on the front of the machine turning on. Then attach the nebulizer to the makeup gas inlet and attach the nebulizer to the liquid introduction tubing. Insert the end of the nebulizer into the white opening in the heating mantle on the RFG controller tab of the instrument setup window.Click start plasma, then click okay. After startup has finished, click okay again in the popup window. Now turn on the syringe pump.When refilling the syringe, press the stop button rather than the pause button to reset the volume counter Before calibrating the mass cytometer, be sure that it has warmed up for 20 minutes. Once the machine has stabilized, 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. Then select the isotopes in the DVS sciences tuning solution listed in the manual and click save. Next, click the isotopes per reading button to open the masses per reading window.Select pulses count and rescale the y axis by entering a new number and then pressing enter. Now fill a green one milliliter norm inject syringe with tuning solution. Turn the sample port selector to load.Inject 450 microliters of tuning solution, and then turn the selector to inject. Wait until the tuning solution isotopes start appearing as streaks in this display. The signals in the time of flight, 9, 400 to 9, 800 region are due to xenon isotopes in the argon gas.Now return to the masses per reading window and click the green play button. Each colored line represents selected isotopes from the analytes tab. When the isotope levels are steady, tune the current select current from the dropdown menu in the parameter tab of the data acquisition settings window.In the start window, enter zero for finish, enter 10. For step value, enter 0.5, and for settling time, enter 200. Then click save.Then click back on the masses per reading window and click play. After noting where the signals of the isotopes peak, go to the DAC channel setup tab of the instrument setup window. Scroll down to current and enter the isotope peak value.Click set actual current value, and then save. Next, change the dropdown menu to make up gas. Enter 0.55 in the start window, 0.95 in the end window, 0.05 in the step value window and 200 in the settling time window.Click save. After returning to the masses per reading window and clicking play again, the x axis will now display the makeup gas flow rate. Take note of the values where the signals of the isotopes peak and observe the rapid increase of the GD 1 55 signal near the end.Then return to the DAC channel setup tab. Scroll down to makeup, gas, and enter the new isotope peak value. Click set actual current value.Then click save again to record the ratio of desired isotope versus oxide. First, make a second 450 microliter injection of tuning solution. Then on the parameter tab, select time from the dropdown menu and enter zero in the start window, 10 in the end window, one in the step value window, and 200 in the settling window.After clicking play in the masses per reading window, use the cursor to read the higher value for TM 1 69 or TB 1 59, and also the GD 1 55 value. Now inject three milliliters of Milli Q water through the sample loop, followed by 500 microliters of DVS wash solution. In the mass calibration tab, click run to monitor the progress of the cleaning wash until the intensity of the streaks begins to decrease.Then follow with three milliliters of milli cube water and monitor until the streaks are down to background levels. Begin by entering the sample isotopes just demonstrated for the tuning solution isotopes, and then click save. Next, open the acquisition window and change the acquisition settings as needed for the sample volume.For example, a 450 microliter sample will fill the sample loop and take 600 seconds to run to allow for delays in the acquisition of the cell data. Set the acquisition delay to at least 30 seconds and the detector stability delay to at least 20 seconds before running. Wash the cell samples at least twice with milli cube water, and then resuspend the sample at a maximum of 10 to the six cells per milliliter and filter the resulting cell solution through a 35 micrometer cell strainer.To remove any aggregates. Now enter a new file name for the current sample. Note that all the files must be saved to the E drive.Then switch the sample loop valve to load, inject the sample, switch the sample loop valve back to inject, and then click run. In the acquisition window. The cells will be represented in the snapshot window as horizontal collections of marks in each vertical isotope channel and will exhibit the best resolution around 300 to 500 cells per second as demonstrated here.When the data acquisition of the sample is finished, a pop-up window will appear. Click okay to confirm completion of the sample run, and then inject at least 500 microliters of MQ water between each sample to help minimize any sample carryover to clean the machine After use, inject 450 microliters of wash solution into the sample loop. Monitor the release of free metal and then flush the sample loop with three milliliters of milli Q water monitoring the wash until background streak levels are achieved.Then to shut down the machine, select the RFG controller tab of the instrument setup window and click stop plasma. When the procedure is complete, click okay in the popup window, recommending the removal of the nebulizer under the card cage tab. Turn off the heater, close the valve on the argonne supply, and then carefully pull backwards on the nebulizer to remove it from the spray chamber.Next, unscrew the sample introduction tubing and place it aside. Unscrew the makeup gas introduction connection until it loosens slightly, and then pull off the nebulizer. Finally, using the same syringe and tubing as demonstrated earlier, clean both ports of the nebulizer with 5%Citron and store the nebulizer covered in 5%Citron until the next use.Adequate washing of the mass cytometer with UE water and DVS wash solution will reduce the levels of metal absorption to the tubing and to other parts of the machine helping to reduce any background as seen in these graphs. Improper tuning represented by the magenta line significantly increased the oxide formation at m plus 16 compared to the properly tuned sample as demonstrated by the dark blue line. This has the effect of slightly decreasing the desired signals at m and significantly increasing the undesired background interference at m plus 16.Proper washing and proper dilution of the samples in Milli Q water will ensure minimal background and the greatest resolution of cells while balancing the need for speed. Similar to the data from a representative experiment shown here in this experiment, polystyrene beads containing natural abundance lanthanum 1 39 ODIUM one 40, TERBIUM 1 59, DIUM 1 69 and LUTETIUM 1 75 were run in the cell acquisition mode. The data was analyzed using FlowJo and debris from any broken beads was gated out.The properly tuned sample was determined to be at the 0.74 liters per minute flow rate as represented with the dark blue line. Higher signal intensities were observed at this flow rate than at the lower flow rates, and this properly tuned sample was observed to have higher signal at the M metal masses and less signal at the m plus 16 oxide masses than the improperly tuned signals. A closeup of the signal at M Mass LANTHANUM 1 39 and THULIUM 1 69 is shown here.Note that the makeup gas flow rate affects the LANTHANUM 1 39 signal intensity more than thulium 1 69 signal intensity. These graphs show the signal at m plus 16 oxide masses 1 55 and 180 5 corresponding to the LANTHANUM 1 39 plus oh 16 and thulium 1 69 plus oh 16 oxides respectively. Note that the makeup gas flow rate increases the mass 1 55 signal much more than the mass 180 5 signal as lanthanum 1 39 is more easily oxidized than thulium 1 69.The m and m plus 16 signal intensities as a function of the makeup gas flow rate are depicted here. The filled icons represent the MASS M.While the open icons represent the mass M plus 16, the colors of the lines correspond to the respective mass M.Note that the LANTHANUM 1 39 and price ODIUM 1 41 signal intensities are highly affected by the makeup gas flow rate. Even reaching a point where more mass m plus 16 oxide is present than mass M.After watching this video, you should have a good understanding of how to start up the CyTOF properly tune it, then run your samples, and finally properly clean and shut down the machine at the end of your daily runs.

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Bioengineering

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