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

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.