Use of the Pyrimidine Analog, 5-Iodo-2′-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

1. Preparation of IdU stocks

1. Dissolve 5-iodo-2’-deoxyuridine (IdU) in DMSO to a concentration of 50 mM. Sterile filter, aliquot into 10-50 µL tubes, and store at -80 °C
2. Remove IdU from the freezer, and thaw at room temperature. Dilute IdU in RPMI-1640 to make a working solution at a final concentration of 1 mM. Pipette up and down or vortex to mix.
   1. Typically, dilute the concentrated IdU into the media in which the cells are being cultured (e.g. DMEM, IMDM, etc.) or have diluted it into PBS for addition directly to peripheral blood or bone marrow aspirate samples. This pre-dilution step facilitates mixing of the DMSO with the aqueous media of the cells of interest.
      NOTE: The final concentration of IdU during incubation should be 10 µM; a solution of 1 mM will can be added at a ratio of 10 µL to every 1 mL of media.

2. IdU incubation and sample preservation

1. Maintain samples in a humidified 37 °C incubator. Remove the sample from the incubator and move the sample into a biosafety hood.
2. Add 10 µL of 1 mM IdU to every 1 mL of sample.
   1. For a 6-well plate, add 30 µL of 1 mM IdU to the 3 mL of culture media in each well. IdU can also be used directly in bone marrow aspirates as well as murine studies⁴.
3. Place the sample back into the incubator at 37 °C for 10-15 min. Maintain cells under the optimal growth conditions of interest during IdU exposure in order to get the most accurate measurement of S-phase.
4. After the IdU incubation, remove the sample and transfer to a conical tube.
5. Spin the sample at 400 x g for 10 min at room temperature.
6. Aspirate the supernatant and resuspend in 200 µL of PBS.
   1. If needed, perform a live/dead stain (using cisplatin) at this step to mark dead cells before fixation and freezing.
      NOTE: Rhodium live/dead staining does not perform well after methanol permeabilization, so it is not recommended for use in cell cycle analyses.
7. Add 18.75 µL of 16% paraformaldehyde (PFA) to the PBS for a final concentration of 1.5% PFA. Incubate at room temperature for 10 min. Spin the sample down at 400 x g for 10 min at room temperature.
8. Aspirate the PBS/PFA solution and re-suspend the sample in 500 µL of Cell Staining Media (CSM; 1x PBS with 0.5% BSA and 0.02% sodium azide) + 10% DMSO prior to freezing.
   1. If using commercial Proteomic Stabilizer, add 280 µL of Proteomic Stabilizer to sample re-suspended in 200 µL of PBS (1:1.4). Incubate samples at room temperature for 10 min and then place directly into the -80 °C.
      NOTE: IdU has been shown to incorporate effectively within 10-15 min incubation at 37 °C. IdU incubations longer than 10-15 min will progressively reduce resolution of the S and G2-phase populations, as IdU-labeled cells leave S phase and progress to G2 or M phase. We have also observed that long-term incubation with IdU can cause cell death and cell cycle artifacts. The subsequent cell processing and antibody staining following IdU incorporation is sufficient to wash away residual IdU that was not incorporated into S-phase cells. We have not observed significant Iodine background when using the in vitro protocol described here; however, we have very rarely observed iodine contamination in clinical samples. This may occur from medical procedures, such as iodine contrast in a CT scan, or from iodine-containing pharmaceuticals. Should large amounts of IdU background be observed, the sample should not be run to avoid damage to the mass cytometer’s detector.

3. Staining samples for mass cytometry

1. Remove the samples from the -80 °C and allow to thaw before surface staining.
   1. If using the SmartTube method of fixation, thaw the samples at 0-4 °C to avoid additional fixation as the samples warm up.
2. After the samples are thawed, transfer approximately 1-2 million cells into a 5 mL FACS tube.
3. Centrifuge the FACS tube at 600 x g for 5 min, and fill the FACS tube with cell staining media (CSM) to wash the cells. Repeat one additional time.
   1. If cells are known to stick together, add 400 U/mL of heparin to CSM washes in order to prevent cell to cell contact but this is not strictly necessary.
4. Incubate the cells with FC-blocking agent, 5 µL of the agent per 100 µL of cells, for 10 min at room temperature.
5. Prepare a mixture of antibodies that will stain the surface, or extracellular portion, of the cells. The total staining mixture will amount to 100 µL per 1-2 million cells in each test. The staining mixture will be balanced accordingly with CSM and heparin in the cocktail and FACS tube.
   NOTE: Addition of CSM at this step will also reduce nonspecific staining artifacts⁸. This staining mixture is entirely dependent on targets of interest and surface phenotype (e.g., a study involving T-cells will use a surface mixture of CD45, CD3, CD4, CD8, etc.). A detailed protocol of sample processing and staining can be found in Behbehani and McCarthy et al.^9,10.
6. Add the surface staining mixture to the cells and incubate at room temperature with continuous shaking for 30-60 min.
7. After staining, fill the FACS tube with CSM, and spin down at 600 x g for 5 min.
8. Wash two more times with CSM, spinning the sample at 600 x g for 5 min and aspirating each CSM wash.
9. Fix the extracellular antibodies by adding 1 mL of PBS with 10% CSM and 1.5% PFA.
10. Fill the FACS tube containing the PBS/CSM/PFA mixture with CSM. Spin down at 600 x g for 5 min and aspirate the supernatant.
11. Add methanol at -20 °C.
12. Vortex the sample for 1-2 min to achieve a single cell suspension and verify that all cell clumps have been re-suspended.
13. While the sample is vortexing slowly, rapidly add 1 mL of ice-cold methanol using a 1,000 µL pipette with a filter tip.
14. Hold the FACs tube up to the light and make sure there are no visible clumps; cloudiness is to be expected. Any clumps will render the sample unusable for subsequent MCM analysis.
15. Store the sample at -20 °C for 10-20 min.
16. Prepare the intracellular staining mixture during this time. The intracellular staining mixture will be dependent on targets of interest. For cell cycle analysis, include CyclinB1, pRb, Ki67, and pHH3 in this staining mixture, but other intracellular markers can be added as needed.
17. After 10-20 min at -20 °C, remove the sample, add 1.5 mL of PBS and fill the remainder with CSM.
18. Centrifuge the sample 600 x g for 5 min, and aspirate the supernatant.
19. Wash two more times with CSM, spinning the sample down at 600 x g for 5 min and aspirating the supernatant each time.
20. After the last CSM wash, centrifuge the sample and leave a residual volume of approximately 50 µL.
21. Add the prepared antibody mixture (typically add 50 µL of antibody staining cocktail to achieve a final staining volume of 100 µL) to the sample and incubate on a shaking platform for 30 to 60 min at room temperature.
22. After staining add CSM and centrifuge at 600 x g for 5 min.
23. Aspirate the CSM, wash again with CSM, spinning at 600 x g for 5 min, aspirating the CSM, then add PBS.
24. After the completion of intracellular staining, place cells into an intercalator solution that fixes the antibodies to the cells and stains the DNA of each cells to enable identification. The intercalator solution contains nonisotopically pure iridium intercalator (pentamethylcyclopentadienyl-Ir(III)-dipyridophenazine) added from the manufacturer’s stock solution at a concentration of 500 µM. Dilute the Iridium stock 1:4000 in a solution of PBS and 1.5% PFA. Add the iridium intercalator solution at 100-200 µL per million cells in order to stain evenly and prevent overstaining.
    NOTE: The iridium in this intercalator solution is intended to identify cells for singlet gating, it should not be used for live/dead stains. If live/dead stains are desired they need to be performed before fixation as noted above and in McCarthy et al.⁹.
25. Store the samples in intercalator solution in a 4 °C refrigerator for up to two weeks before sample acquisition on the CyTOF.

4. Mass cytometer operation

NOTE: Mass cytometry operation can be machine specific. It is always advisable to check the CyTOF user’s manual before operation. Additionally, there are currently two JoVE articles dealing with machine start up and maintenance^9,11.

1. Check the nebulizer for any clogs, cracks, and other irregularities before operating the mass cytometer.
2. Connect the nebulizer to the cytometer and begin the warmup procedure. Do not start the mass cytometer without the nebulizer in place.
3. Run water through the sample lines once the mass cytometer has finished warming up. The spray chamber needs to reach approximately 200 °C before performing tuning or sample analysis.
4. Run water for 5-10 min. After 5-10 min, load the tuning solution and select the tuning manager. The tuning solution is a solution containing fixed concentrations of metals and used to optimize the mass cytometer before sample acquisition
5. In the tuning manager, select Visualizar once the tuning solution has reached a steady state hit record to begin the automated tuning process.
6. Once tuning is finished load the sampler with water and allow water to run through the sample lines during sample processing. Detailed protocol for daily cytometer operation and tuning can be found at Leipold¹¹.
7. Occasionally, the automated tuning will not tune to optimal machine performance. Repeat the tuning procedure to correct this.
8. Wash the sample with CSM once and with pure deionized water twice before sample acquisition. Washing with water is important to remove residual salt from the PBS/CSM.
9. Check the sensitivity and sample flow using manufacturer supplied equilibration beads, polystyrene beads loaded with known metal concentrations.
10. Change the acquisition mode from tuning to event capture mode. Set the time limit to stop acquisition at 120 s. Wait 45 s before selecting Record.
11. The mass cytometer will stop sample acquisition automatically after 120 seconds. Use the rain plot viewer to check Eu151 and Eu153 intensity.
12. Dilute equilibration beads in pure deionized water at a 1:20 ratio.
13. Before sample acquisition check the experiment manager. Use the experiment manager to assign names to channels and to add channels to be recorded.
    1. Make sure the 127-I channel is added if using IdU.
    2. Note that it is essential to set the mass cytometer to measure the needed parameters (e.g., IdU) prior to sample acquisition. If channels are not selected in advance, data will not be collected from any unselected channel and cannot be recovered.
14. Dilute the cells to a concentration of approximately 1-2 x 10⁶/mL using the 1:20 pure deionized water and equilibration bead mixture. Pass the cells through the filter topped FACS tube in order to remove any residual clumps.
15. Load the sample and change the acquisition time.
16. Press Visualizar and wait for event count per second to stabilize.
    1. Do not run events in excess of 400 events per second, this will lead to significant amounts of doublets and debris. We typically collect at least 20,000 to 50,000 cell events, but the optimal number will depend on the experimental design. Staining of up to 2 million cells will typically yield 300,000 to 400,000 cell events. Note that not all events will be cells (there will be debris and bead events included in the event count).
17. Once sample acquisition is done load a washing solution, start sample induction and run for 5-10 minutes. After 5-10 minutes stop sample induction and run water for 10-20 minutes. Wash solution is a weak solution of hydrofluoric acid designed to strip residual metal from the sample lines.
18. Shut down the mass cytometer and remove the nebulizer. The nebulizer will be hot, take care during handling.

5. Data analysis

1. In order to remove beads and also to correct for signal drift during sample acquisition, normalize the FCS files using Fluidigm software or the application developed by Finck¹².
2. Upload the FCS to Cytobank or other flow cytometry analysis software. FCS files can be used in any compatible software, for the purposes of this protocol all gating and further analysis has been done in Cytobank¹³.
3. Before cell cycle gates can be drawn, exclude any doublets or cell debris from downstream analysis, this can be done by using the biaxial plot of Event Length vs 191-Ir (Figure 1a). Cells will form a distinct, bright population Ir^high that can be used to exclude doublets and debris. This is the singlet gate. This gating method typically removes about 50-60% of doublet cell events, so additional strategies may be required to remove remaining doublet cell events.
   1. Change the event length scale (minimum and maximum) to make the cells appear more prominent to aid in singlet gating.
   2. Further remove doublets and debris by using Gaussian parameters, Residual and Offset. A higher Residual with lower Offset is also debris and doublets, and gating around this population can further remove doublets and debris (Figure 1b,c).
4. S-phase gating – S-phase is the easiest gate to draw but also the most important. Draw this gate using a biaxial plot of IdU vs pRb, Ki67, or cyclinB1. S-phase IdU⁺ cells will form a distinct population when looking at these biaxial plots (Figure 2b).
5. G0/G1-phase, G2/M-phase gating – Establish the G0/G1 and G2/M phase gates on the IdU vs CyclinB1 plot and the use of IdU incorporation is crucial to establish the boundary between the G0/G1 and G2/M phase gates. G0/G1-phase will be CyclinB1^low/IdU^– and G2/M-phase will be CyclinB1^high/IdU^–. Good CyclinB1 staining will show a natural population between the G0-G1 and G2-M populations; however, this will vary across sample and cell types under experimental conditions. In experimental conditions where the cell cycle distribution may be affected and there is less separation between the CyclinB1 G0/G1-phase and G2/M-phase utilizing the S-phase will allow consistent gating for particular cell types in each specific experiment. This method is detailed below.
   1. Plot only the S-phase cells on the CyclinB1 vs IdU to help establish the separation between G0/G1-phase and G2/M-phase. Draw a gate on the CyclinB1^high population and adjust until approximately the top 5% of the S-phase population is inside the gate (Figure 2c). This establishes the breakpoint between the G0/G1 and G2/M phase gates (Figure 2e,f). The active population will be changed to the population of interest and the portion residing inside the previous gate will be the G2/M-phase population while the remainder will be the G0/G1-phase population.
6. G0-phase gating – Establish the G0-phase on the pRb vs IdU plot. The G0-phase will be represented by a pRb^low/IdU^– population. The active cycling population will have high expression of pRb and IdU incorporation, the G0-phase gate can be drawn on this boundary as it typically expresses at two distinct populations (Figure 2g,i).
   1. Define the G0-phase by making the S-phase population drawn previously (Figure 2b) the active population and drawing a gate incorporating the top 90-100% of the pRb^high population. This is the pRb+ cycling population (Figure 2i), the pRb^low population outside this gate is the G0 population (Figure 2j).
   2. If pRb is unavailable or not able to be recorded, use Ki67 vs IdU to establish the G0-phase population. Drawing a gate representing the majority of the S-phase population and using that gate as the boundary for the Ki67 vs IdU the remainder of the population will be the G0-phase (Figure 3a).
7. M-phase gating – Establish the M-phase in the IdU vs pH3 biaxial plot. The M-phase represents a very small fraction of cells and is gated on the pH3^high population (Figure 2d).
8. IdU incorporation failed or was not possible – If IdU is unavailable, define cell cycling and not cycling fractions using Ki67 and pRb. Ki67 and pRb, in normal conditions, form two distinct populations a Ki67^high/pRb^high and a Ki67^low/pRb^low. The double positive population represents the active cycling population, correlating to G1-phase, S-phase, G2-phase, and M-phase. The double low population represents the not cycling population, correlating to the G0-phase (Figure 3b).
   NOTE: It is not possible to delineate each individual phase using the Ki67 vs pRb but experimental effects on the relative cycling/not cycling populations can be determined.
9. Cell cycle analysis – Once the gates have been established, export the numerical values from the gates for further analysis. The percentages in each cycle can be achieved by subtracting the single populations from the combined populations. The G0-phase, drawn on the pRb^lowIdU^low, gate percentage can be subtracted from the G0/G1-phase, drawn on CyclinB1^lowIdU^neg, to find the G1-phase percentage. Similarly the G2-phase percentage is derived from the subtraction of the M-phase gate from the G2/M-phase gate. This will generate numerical values for each individual cell cycle phase; G0, G1, S, G2, and M. The numerical values generated for each individual cell cycle phase can be used for further analysis such as graphing and statistical analysis.