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-2Ã¢â‚¬Â²-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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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. 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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-2Ã¢â‚¬Â²-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-2ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â²-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 ...

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