Name: use of the pyrimidine analog, 5-iodo-2&prime;-deoxyuridine (idu) with cell cycle markers to establish cell cycle phases in a mass cytometry platform
Uploaded: 2021-10-22T13:00:00
Description: Watch this Scientific Journal Video about Use of the Pyrimidine Analog, 5-Iodo-2â€²-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform at JoVE.com

The authors would like to thank the efforts of Palak Sekhri, Hussam Alkhalaileh, Hsiaochi Chang, and Justin Lyeberger for their experimental support. This work was supported by the Pelotonia Fellowship Program. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the Pelotonia Fellowship Program.&#34;

1. Preparation of IdU stocks
<ol>
	<li>Dissolve 5-iodo-2&#8217;-deoxyuridine (IdU) in DMSO to a concentration of 50 mM. Sterile filter, aliquot into 10-50 &#181;L tubes, and store at -80 &#176;C</li>
	<li>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.
	<ol>
		<li>Typically, dilute the concentrated IdU into the media in which the cells are being cultured (e.g. DMEM, IMDM, etc.) or have d...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Cytometric+Functional+Profiling+of+Acute+Myeloid+Leukemia+Defines+Cell-Cycle+and+Immunophenotypic+Properties+That+Correlate+with+Known+Responses+to+Therapy.">Mass Cytometric Functional Profiling of Acute Myeloid Leukemia Defines Cell-Cycle and Immunophenotypic Properties That Correlate with Known Responses to Therapy.</a> Cancer Discovery. 5 (9), 988-1003 (2015).</li><li>Kimmey, S. C., Borges, L., Baskar, R., Bendall, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+analysis+of+tri-molecular+biosynthesis+with+cell+identity+and+function+in+single+cells.">Parallel analysis of tri-molecular biosynthesis with cell identity and function in single cells.</a> Nature Communications. 10 (1), 1185 (2019).</li><li>Rabinovitch, P. S., Kubbies, M., Chen, Y. C., Schindler, D., Hoehn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU-Hoechst+flow+cytometry:+a+unique+tool+for+quantitative+cell+cycle+analysis.">BrdU-Hoechst flow cytometry: a unique tool for quantitative cell cycle analysis.</a> Experimental Cell Research. 174 (2), 309-318 (1988).</li><li>Rahman, A. H., Tordesillas, L., Berin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+reduces+nonspecific+eosinophil+staining+artifacts+in+mass+cytometry+experiments.">Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments.</a> Cytometry A. 89 (6), 601-607 (2016).</li><li>McCarthy, R. L., Duncan, A. D., Barton, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+Preparation+for+Mass+Cytometry+Analysis.">Sample Preparation for Mass Cytometry Analysis.</a> Journal of Visualized Experiments. (122), e54394 (2017).</li><li>Behbehani, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping+by+Mass+Cytometry.">Immunophenotyping by Mass Cytometry.</a> Methods in Molecular Biology. 2032, 31-51 (2019).</li><li>Leipold, M. D., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> Journal of Visualized Experiments. (69), e4398 (2012).</li><li>Finck, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+mass+cytometry+data+with+bead+standards.">Normalization of mass cytometry data with bead standards.</a> Cytometry A. 83 (5), 483-494 (2013).</li><li>Kotecha, N., Krutzik, P. O., Irish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+analysis+and+publication+of+flow+cytometry+experiments.">Web-based analysis and publication of flow cytometry experiments.</a> Current Protocols in Cytometry. , 17 (2010).</li><li>Devine, R. D., Sekhri, P., Behbehani, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+storage+time+and+temperature+on+cell+cycle+analysis+by+mass+cytometry.">Effect of storage time and temperature on cell cycle analysis by mass cytometry.</a> Cytometry A. 93 (11), 1141-1149 (2018).</li><li>Campos, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+immunological+markers+on+leukemic+cells+before+and+after+cryopreservation+and+thawing.">Expression of immunological markers on leukemic cells before and after cryopreservation and thawing.</a> Cryobiology. 25 (1), 18-22 (1988).</li><li>Kadic, E., Moniz, R. J., Huo, Y., Chi, A., Kariv, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cryopreservation+on+delineation+of+immune+cell+subpopulations+in+tumor+specimens+as+determinated+by+multiparametric+single+cell+mass+cytometry+analysis.">Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.</a> BMC Immunology. 18 (1), 6 (2017).</li><li>Shabihkhani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+procurement,+storage,+and+quality+assurance+of+frozen+blood+and+tissue+biospecimens+in+pathology,+biorepository,+and+biobank+settings.">The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings.</a> Clinical Biochemistry. 47 (4-5), 258-266 (2014).</li></ol>

The examples presented here demonstrate how to use an MCM platform to analyze cell cycle distribution. It has also been demonstrated that cell cycle analysis is sensitive to experimental conditions such as time and temperature, which is an important consideration researchers must take when considering MCM for their cell cycle analysis14. Samples left in storage for a short period of time, no longer than an hour, will have IdU incorporation comparable to their normal state. Samples in a closed syst...

Mass cytometry enables detection of approximately 40 parameters by taking advantage of the high resolution and quantitative nature of mass spectroscopy. Metal-labeled antibodies are used instead of fluorophore conjugated antibodies that allow for a higher number of channels and produce minimal spillover1,2. MCM has advantages and disadvantages in regard to cell cycle analysis in comparison to flow cytometry. One major advantage of MCM is that the large number of parameters enables the simultaneous measurement of cell cycle state across a large number of immunophenotypically distinct T-cell types in highly heterogeneous samples. MCM has been successfully used to measure the cell cycle state during normal hematopoiesis in human bone marrow3 and transgenic murine models of telomerase deficiency4. Analysis of cell cycle state in acute myeloid leukemia (AML) showed that cell cycle correlated to known responses to clinical therapies, providing an in vivo insight into functional characteristics that can inform therapy selections5. A second advantage of mass cytometric cell cycle analysis is the ability to measure a large number of other functional markers that may be correlated with cell cycle state. Recent work has been able to correlate protein and RNA synthesis with cell cycle state through the use of IdU and metal tagged antibodies to BRU and rRNA6. This kind of highly parametric analysis measuring cell cycle state across numerous populations in a continuum of differentiation would be nearly impossible with current flow cytometry technology. The major disadvantage of MCM is the lack of comparable DNA or RNA stains as those used in fluorescent flow cytometry (e.g., DAPI, Hoechst, Pyronin Y, etc.). Fluorescent dyes can give relatively precise measurements of DNA and RNA content, but this precision is only possible due to the changes in the fluorescent properties of these dyes that occur upon intercalation between nucleotide bases. MCM analysis is thus unable to measure DNA or RNA content with similar precision. Instead, mass cytometric cell cycle analysis relies on measurements of proteins related to cell cycle state such as cyclin B1, phosphorylated retinoblastoma protein (pRb), and phosphorylated Histone H3 (pHH3) combined with direct measurement of the iodine atom from IdU incorporation into S-phase cells. These two measurement approaches yield highly similar results during normal cellular proliferation, but can potentially be discordant when cell cycle progression is disrupted.
Measurement of the number of cells in each cell cycle phase is important in understanding normal cell cycle development as well as cell cycle disruption, which is commonly observed in cancers and immunological diseases. MCM provides reliable measurement of extracellular and intracellular factors using metal-tagged antibodies; however, measurement of the S-phase was limited as the iridium-based DNA intercalator was unable to differentiate between 2N and 4N DNA. In order to define cell cycle phases, Behbehani developed a method that utilizes IdU with a mass of 127, which falls within the range of the mass cytometer and allows direct measurement of cells in S-phase3. This direct measurement circumvents the need for secondary antibodies or use of DNA denaturing agents such as acid or DNase. In conjunction with intracellular cycling markers, it allows high resolution of cell cycle distribution in experimental models.
This protocol adapts cell cycle measurements from common flow cytometry protocols for MCM. Our methods provide a convenient and simple way to include cell cycle parameters. IdU incorporation of in vitro samples requires only 10 to 15 minutes of incubation at 37 &#176;C, which is shorter than most BrdU staining protocols that recommend incubation times of several hours3,7. IdU and BrdU incorporated samples can be fixed using a proteomic stabilizer and then stored for some time in a -80 &#176;C freezer. This allows large numbers of IdU stained samples to be archived for batch analysis without reduction in sample quality.

<table>
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A3059</td>
			<td>Component of CSM</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-217-420</td>
			<td>Sample centrifugation</td>
		</tr>
		<tr>
			<td>Cleaved-PARP (D214)</td>
			<td>BD Biosciences</td>
			<td>F21-852</td>
			<td>Identification of apoptotic cells</td>
		</tr>
		<tr>
			<td>Cyclin B1</td>
			<td>BD Biosciences</td>
			<td>GNS-1</td>
			<td>G2 Resolution</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>Cryopreservative</td>
		</tr>
		<tr>
			<td>EQ Four Element Calibration Beads</td>
			<td>Fluidigm</td>
			<td>201078</td>
			<td>Internal metal standard for CyTOF performance</td>
		</tr>
		<tr>
			<td>FACS Tube w/ mesh strainer</td>
			<td>Corning</td>
			<td>08-771-23</td>
			<td>Cell strainer to remove clumps/debris before CyTOF run</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td>Cell culture growth supplement</td>
		</tr>
		<tr>
			<td>Helios</td>
			<td>Fluidigm</td>
			<td></td>
			<td>CyTOF System/Platform</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>Staining additive to prevent non-specific staining</td>
		</tr>
		<tr>
			<td>IdU (5-Iodo-2&#8242;-deoxyuridine)</td>
			<td>Sigma</td>
			<td>I7125</td>
			<td>Incorporates in S-phase</td>
		</tr>
		<tr>
			<td>Ki-67</td>
			<td>eBiosciences</td>
			<td>SolA15</td>
			<td>Confirmation of G0/G1</td>
		</tr>
		<tr>
			<td>MaxPar Multi Label Kit</td>
			<td>Fluidigm</td>
			<td>201300</td>
			<td>Metal labeling kit, attaches metals to antibodies</td>
		</tr>
		<tr>
			<td>Microplate Shaker</td>
			<td>Thermo Scientific</td>
			<td>88880023</td>
			<td>Mixing samples during staining</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>pentamethylcyclopentadienyl-Ir(III)-dipyridophenazine</td>
			<td>Fluidigm</td>
			<td>201192</td>
			<td>Cell identification during CyTOF acquisition</td>
		</tr>
		<tr>
			<td>p-H2AX (S139)</td>
			<td>Millipore</td>
			<td>JBW301</td>
			<td>Detection of DNA damage</td>
		</tr>
		<tr>
			<td>p-HH3 (S28)</td>
			<td>Biolegend</td>
			<td>HTA28</td>
			<td>M-phase Resolution</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Wash solution for cell culture and component of fixative solution</td>
		</tr>
		<tr>
			<td>p-Rb (S807/811)</td>
			<td>BD Biosciences</td>
			<td>J112906</td>
			<td>G0/G1 Resolution</td>
		</tr>
		<tr>
			<td>Proteomic Stabilizer</td>
			<td>SmartTube Inc</td>
			<td>PROT1</td>
			<td>Sample fixative</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td>Cell culture growth medium</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Acros Organics</td>
			<td>AC447810250</td>
			<td>Component of CSM/Antibody buffer, biocide</td>
		</tr>
	</tbody>
</table>

use of the pyrimidine analog, 5-iodo-2&prime;-deoxyuridine (idu) with cell cycle markers to establish cell cycle phases in a mass cytometry platform

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the cell cycle is a feature of a number of diseases, including cancer. Study of the cell cycle necessitates the ability to define the number of cells in each portion of cell cycle progression as well as to clearly delineate between each cell cycle phase. The advent of mass cytometry (MCM) provides tremendous potential for high throughput single cell analysis through direct measurements of elemental isotopes, and the development of a method to measure the cell cycle state by MCM further extends the utility of MCM. Here we describe a method that directly measures 5-iodo-2&#8242;-deoxyuridine (IdU), similar to 5-bromo-2&#180;-deoxyuridine (BrdU), in an MCM system. Use of this IdU-based MCM provides several advantages. First, IdU is rapidly incorporated into DNA during its synthesis, allowing reliable measurement of cells in the S-phase with incubations as short as 10-15 minutes. Second, IdU is measured without the need for secondary antibodies or the need for DNA degradation. Third, IdU staining can be easily combined with measurement of cyclin B1, phosphorylated retinoblastoma protein (pRb), and phosphorylated histone H3 (pHH3), which collectively provides clear delineation of the five cell cycle phases. Combination of these cell cycle markers with the high number of parameters possible with MCM allow combination with numerous other metrics.

Utilizing HL-60 cells and a human bone marrow aspirate it is possible to show how experimental conditions can affect cell cycle distribution and analysis. First, the gating strategy must be established to demonstrate how the cell cycle phases are derived. In Figure 1 we show the establishment of the singlet gate, which is important in separating cellular debris and doublets, establishing a single cell population. For cell lines the singlet gate is all that is needed to move onto cell cycle a...

Watch this Scientific Journal Video about Use of the Pyrimidine Analog, 5-Iodo-2â€²-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform at JoVE.com

Use of the Pyrimidine Analog, 5-Iodo-2&#8242;-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

This protocol adapts cell cycle measurements for use in a mass cytometry platform. With the multi-parameter capabilities of mass cytometry, direct measurement of iodine incorporation allows identification of cells in S-phase while intracellular cycling markers enable characterization of each cell cycle state in a range of experimental conditions.

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

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the ...

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.

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