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

This protocol allows us to examine the cell cycle at the single cell level using the mass cytometer, and this enables several novel research and clinical studies to be performed. The main advantage of this technique is its simplicity. IdU is added directly to the cells and there is no need for further treatment or antibodies.

We've used this technique to investigate the cell cycle state in patients with acute myeloid leukemia within the stem and progenitor cell compartments. And this demonstrated that the cell cycle properties of these specific cell types may correlate with the clinical outcome of patients with these diseases. This technique can be useful for understanding growth properties of rare cancer cells and understanding the proliferation of immune cell subsets.

Before and after adding IdU to the cell samples, maintain the cells in the growth conditions of interest. In this case, a humidified 37 degrees Celsius incubator at 5%CO2 to label the cells with IdU, dilute the concentrated IdU stock solution one to 50 into the growth media of the cells. Then transfer the samples into a biosafety hood and add 10 microliters of one millimolar IdU to every one milliliter of cells.

Return the cells to the incubator for 10 to 15 minutes before transferring the samples to individual conical tubes. Collect the cells by centrifugation in 15 milliliter conical tubes and resuspend the pellets in 200 microliters of PBS per tube. If needed, label the cells with an appropriate live/dead stain to mark the dead cells.

Following live/dead stain, quenched the cells with media washed with CSM before fixing the cells with 25 microliters of 16%paraformaldehyde. After a 10 minute incubation at room temperature, collect the cells by centrifugation. Add five to 15 milliliters of CSM and centrifuge again.

Resuspend the pellets in 500 microliters of cell staining medium supplemented with 10%dimethyl sulfoxide. Then transfer the cells into smaller tubes to store them in negative 80 degrees Celsius. To normalize the for their cell cycle analysis, import the files into an appropriate flow cytometry analysis software program and create a biaxial plot of the event length versus 191 iridium.

The cells will form a distinct bright iridium high population. Change the event length scale to make the cells appear more prominent. Then create a singlet gate to exclude 50 to 60%of the doublets and debris and set the residual and offset Gaussian parameters to remove any remaining doublets and debris.

To set the S-phase gate, create a biaxial plot of IdU versus a proliferation marker. The S-phase IdU positive cells will form a distinct population. For G0/G1, and G2/M gating, establish the gates on an IdU versus Cyclin B1 plot.

The G0/G1 phase will be Cyclin B1 low, IdU negative, and the G2/M phase will be Cyclin B1 high, IdU negative. To gate for specific cell types, plot only the S-phase cells on the Cyclin B1 versus IdU graph to help establish the separation between G0/G1 phase and G2/M phase cells. Draw a gate around the Cyclin B1 high population.

In some cases, this can be difficult to discern but the level of Cyclin B1 of approximately top 5%of the S-phase population is typically the breakpoint between the G0/G1 and G2/M phase gates. The cells residing within the previous gate will be the G2/M phase population, while the remainder will be the G0/G1 phase population. To establish the G0 phase population, create a phosphorylated retinoblastoma protein versus IdU plot.

The G0 phase will be represented by a phosphorylated retinoblastoma protein low, IdU negative population. As the active cycling population will demonstrate a high phosphorylated retinoblastoma protein and IdU incorporation, the G0 phase gate can be drawn on this boundary as it typically expresses at two distinct populations. If the G0 gate is difficult to define, make the S-phase population the active population and draw a gate incorporating the top 90 to 100%of the phosphorylated retinoblastoma protein high population.

The phosphorylated retinoblastoma protein low population outside of the gate is typically the G0 population, while the G0/G1 cells with pRb levels that fall in this gate are G1 cells. For M-phase gating, create an IdU versus phosphorylated histone H3 by axial plot. The M-phase phase will be observed as the very small fraction of cells within the phosphorylated histone H3 population.

The cells in the G2/M population with lower levels of pHH3 are the G2 cells. If IdU incorporation failed or was not possible, define the cycling and not cycling cell fractions using Ki67 and phosphorylated retinoblastoma protein. The double positive population will represent the active cycling population correlating to the G1, S, G2, and M-phases.

The the double low population will represent the not cycling population correlating to the G0 phase. Once all of the gates have been established, export the numerical values from the gates. The percentages in each cycle can be achieved by subtracting the single populations from the combined populations to generate numerical values for each individual cell cycle phase for subsequent graphing and statistical analyze.

The establishment of the singlet gate is important for separating the cellular debris and doublets to allow isolation of the single cell population. Cell cycle analysis of the single cells can then be performed. IdU labeling and thus downstream cell cycle analysis can significantly be affected by time and temperature.

For example, cells that remain too long in enclosed vessels or in transport between locations will have reduced S-phase fractions and that will not be accurate for cell cycle analysis. Cell samples held for short periods of an hour or less still demonstrate a normal cell cycle distribution, indicating that a quick transport may not negatively impact the analysis. Cryopreserved cells may require a long equilibration period before the cells return to active cell cycling, which may still not reflect the pre-cryopreservation cell cycle state.

Different cell types may also be differentially affected by maintenance conditions. For example, in this human bone marrow sample, the T-cells demonstrated a reduction in cell numbers compared to monoblasts from the same sample. Another benefit of mass cytometry is the ability to discriminate cells in cell cycle arrest or that have an abnormal cell cycle distribution.

A major advantage of this technique is it is easily compatible with other mass cytometry measurements such as surface phenotyping, chromatin structure, intracellular cytokine stimulate, which can be correlated with cell cycle state. We've used this technique to study immune cell exhaustion, senescence, and to show that the response to chemotherapy in patients with acute leukemia may correlate with the cell cycle properties of the leukemia cells.