Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University
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Leipold, M. D., Maecker, H. T. Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer. J. Vis. Exp. (69), e4398, doi:10.3791/4398 (2012).
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.
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
Figure 1. CyTOF panel lights prior to startup.
Figure 2. CyTOF panel lights after plasma ignited and start-up successfully completed.
2. Daily Calibration of the Mass Cytometer
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. Click here to view larger figure.
Figure 4. Mass calibration tab window, showing vertical streaks for various isotopes in DVS tuning solution. Click here to view larger figure.
Figure 5. Current tuning profile.
Figure 6. Make-up gas tuning profile.
Figure 7. Measurement of tuning solution signal intensities after optimizing Current and Make-up gas.
3. Running Samples
Figure 8. Acquisition window during sample running, showing horizontal rows of spots corresponding to the elements associated with three separate cell events. Click here to view larger figure.
4. Cleaning the Machine After Use
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. Click here to view larger figure.
5. Shutting Down the Machine
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 remove any cells that might get stuck in the machine and thereby minimizing carryover from sample to sample. Third, while oxide formation cannot be completely prevented, proper tuning of the mass cytometer (Figures 5-7) will help optimize total signal at the desired mass M, while minimizing background at the oxide mass M+16. As seen in Figure 10, improper tuning (magenta) significantly increased oxide formation at M+16 compared to the properly tuned sample (dark blue). This has the effect of slightly decreasing desired signal at M, and significantly increasing undesired background interference at M+16.
Finally, adequate washing of the sample in buffers and finally in MilliQ water should reduce metal/antibody background signal to acceptable levels. The MilliQ water washes and resuspension are critical for maintaining steady Current settings throughout the course of several hours of runtime. Proper dilution of the sample in MilliQ water has to be determined on a daily sample basis. However, diluting to ~106 (starting) cells/ml is a good place to start. Based on the first sample of a set, greater or lesser dilution for subsequent samples can be accommodated. Proper washing and proper dilution will ensure minimal background and the greatest resolution of cells, while balancing the need for speed (Figure 8).
Figure 10. Effect of proper tuning on desired signal, and difference in oxide formation between various lanthanide metals. Polystyrene beads containing natural-abundance La139, Pr141, Tb159, Tm169, and Lu175 from DVS Sciences were run in cell acquisition mode. Separate samples of the beads were run at the indicated Make-up gas flow rates (compare with Figure 6). The data was analyzed using FlowJo v9.4.9 for Mac, including gating out debris from broken beads. The properly tuned sample was at 0.74 L/min. This had higher signal intensities than at lower flow rates, and higher signal at "M" metal masses with less signal at the "M+16" oxide masses than the improperly tuned signal. A. Signal at "M" mass La139 and Tm169. Note that La139 signal intensity is affected more than Tm169 by Make-up gas flow rate. B. Signal at "M+16" oxide masses 155 and 185, corresponding to La139+O16 and Tm169+O16 oxides, respectively. There is no actual "Gd155" or "Re185" present in the beads. The oxide signal in the "Gd155" channel is particularly strong, due to La139 being easily oxidized. This represents one of the highest levels of oxide one can expect to encounter. C. Graph of "M" and "M+16" signal intensities as a function of Make-up gas flow rate. Filled icons represent mass "M", while open icons represent mass "M+16". The color of the lines correspond to the respective mass "M". Notice that La139 and Pr141 are highly affected by Make-up gas flow rate, even reaching a point where more mass "M+16" oxide is present than mass "M". Click here to view larger figure.
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 uses polymers with chelating sites for metals as labels for antibodies.1-3 Due to the purity of the metals and the mass resolution of ICP-MS, there is effectively no "spectral overlap." Since most of the elements used are not biologically relevant, there is also little background to cause "autofluoresence." These facts allow the use of more than 30 simultaneous markers within a single experiment.6,7 There is also a wider dynamic range in signal in ICP-MS than in a typical fluorescence experiment.
However, mass cytometry has limitations. It is a destructive technique: cells cannot be recovered for sorting and further experimentation. The mass cytometer has a strict requirement for the presence of metals: if a metal is not present, a cell will not be detected. This is the primary reason for the use of metal-chelating DNA intercalators to ensure correct percent-of-parent statistics, as all cells will be detected regardless of whether any antibody probes bind.3,6-7 Finally, the current cell transmission efficiency of the machine is ~25%, compared to an efficiency of >90% for a fluorescent flow cytometer. Therefore, a larger starting number of cells is often required, particularly for rare cell populations. However, this is usually balanced out by the fact that a single mass cytometer sample often replaces multiple fluorescent samples. Finally, the maximum cell acquisition rate is much slower than standard fluorescent flow cytometers at ~1,000 cells/sec; optimal rate is often half that. Therefore, each sample takes longer to run.
Several papers have recently been published on the staining of various types of cell samples.3,5-7 The purpose of this article is to provide information on the proper use of a CyTOF mass cytometer for running samples. Maintenance of the nebulizer by backflushing with and storage in 5% citranox will minimized build-up of cell debris and metals, thereby reducing the likelihood of clogs. Maintenance of the machine for optimal performance has two parts. First, the proper tuning (Current, Make-up gas) of the machine on at least a daily basis helps ensure proper measurement. Second, adequate washing with MilliQ water between each sample and with Wash solution at the end of runs to remove built-up organic and inorganic debris will help minimize background in general and carryover between samples. Finally, the quality of sample preparation has a great impact on the quality of data acquisition. Multiple washes in buffer and finally in MilliQ water help remove any metal-background from intercalators or nonspecific antibody binding. Proper dilution of the cell sample will help balance optimal resolution between cell events while minimizing total run time per sample. Proper dilution also helps minimize the likelihood of causing a clog in the nebulizer.
Troubleshooting Common Issues
1. The machine doesn't get past "RFG Prepare" step during start-up
Under the Card Cage tab of the Instrument Set-up window, press "Reset" under RFG. Attempt to start the machine. If this doesn't fix the problem, close the software, reopen, press "Reset" again, and attempt to start. If this still doesn't fix the problem, check the RFG generator breaker switch on the left back corner of the machine. If tripped, reposition to "On", hit "Reset", and try to start. If this still doesn't fix the problem, contact DVS.
2. The machine shuts down while running
This can also happen due to large droplets that begin to form when the nebulizer starts to clog. If this error recurs, remove the nebulizer for cleaning, insert a clean nebulizer and restart.
Much less frequently, the thin sample tubing between the sample loop and the nebulizer can clog. This can be diagnosed by anomalously low flow rate of liquid when the tubing is removed from the nebulizer. If this is the case, remove the affected tubing and fittings to be rebuilt with fresh tubing, and replace with a new nebulizer fittings and tubing kit.
3. No isotope streaks (or cell events) visible during cell sample acquisition (Figure 8), or while watching Mass calibration tab window (Figures 3, 4, 9)
Issues 3c and 3d can also be checked by running a sample of the Eu-containing polystyrene calibration beads. They are supplied at approximately 1 million/ml (vortex well before drawing sample!). Therefore, filling the 450 μl sample loop would give approximately 450,000 beads. The CyTOF cell transmission efficiency is machine-dependent, but approximately 15-30%. Therefore, 67,500-135,000 bead events would be expected from that injection. Numbers below that are consistent with a clog in the tubing or nebulizer.
The cell/bead transmission efficiency is something to test occasionally, to note any long-term trends.
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.
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.
|CyTOF mass cytometer||DVS Sciences|
|Wash solution||DVS Sciences||201071||0.05% hydrofluoric acid in water|
|Tuning solution||DVS Sciences||201072||0.5 ppm La, Cs, Tb, Tm, Ir, trace nitric acid|
|Eu-containing polystyrene beads||DVS Sciences||201073||Contains natural-abundance Eu isotopes; beads supplied at approximately 1 million/ml|
|Multi-lanthanide-containing (La/Pr/Tb/Tm/Lu)- polystyrene beads||DVS Sciences||Not yet commercially-available||Contains natural-abundance isotopes of listed lanthanides|
|Nitric acid (concentrated)||Fisher Scientific||A467-500||Optima trace-metal pure ICP-MS grade|
|Polystyrene round-bottom tube with cell-strainer cap-5 ml||BD||352235||used to filter cell samples before injection|
|Norm-Ject tuberkulin syringe-1 ml||Henke Sass Wolf||4010-200V0||silicone-free, latex-free|
|Norm-Ject syringe-3 ml||Henke Sass Wolf||4010.000V0||silicone-free, latex-free|
|MilliQ water||18 MΩ 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).|
|Argon gas||Praxair||AR 5.0UH-T||99.999% Ultra-high purity|