Support for this work came from the Department of Dermatology, the Gates Center for Regenerative Medicine at the University of Colorado and&#160;University of Colorado (UC) Skin Disease Center Morphology and Phenotyping Cores (NIAMS P30 AR057212). The authors acknowledge the UC Cancer Center Flow Cytometry Shared Resource and support grant (NCI P30 CA046934) for the operation of the mass cytometer and are grateful for Karen Helm and Christine Childs at the core for their expert advice on flow and mass cytometric techniques.

The University of Colorado Anschutz Medical Campus&#39; Institutional Animal Care and Use Committee approved the animal experiments described in this protocol.
1. Preparations
<ol>
	<li>Design a metal-tagged antibody panel. Use the free online panel design software12 and include 127IododeoxyUridine (IdU), 164Dy (Dysprosium) labeled anti-CCNB1 (CYCLIN B1), 175Lu (Lutetium) phospho&#160;(p)-HISTONEH3Ser28 (pHH3), and 150<...

<ol>
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The authors have nothing to disclose.

The protocol outlined in this paper can be completed in about 8 h. The end result is a suspension of cells enriched in KCs that can be analyzed for protein expression in a cell cycle-dependent manner. Several previous studies have outlined methods to isolate KCs from human and mouse skin16,25. These studies also include protocols for the isolation of KCs for flow cytometry26. However, a detailed protocol has not been previously described t...

Correlation of&#160;gene expression with cell cycle stages remains a challenge in the analysis of animal models of hyperplastic diseases like cancer. Part of this challenge is the co-detection of proteins of interest (POI) with markers of proliferation. Proliferative cells can be found in various cell cycle phases including G1, S, G2, and M. Ki67 is one of most commonly used markers of proliferation and is expressed in all phases of the cell cycle. It has been extensively used in the analysis of both human and mouse tissues1,2,3. However, like other general proliferation markers, Ki67 does not discern individual cell cycle phases. A more precise approach uses the incorporation of thymidine nucleotide analogs like Bromodeoxyuridine (BrdU) into cells that are actively replicating their genome (i.e., S-phase)4,5. One drawback to the use of nucleotide analogs is the need to administer them to live animals hours before analysis. Ki67 and BrdU are commonly detected on fixed tissue sections by the use of antibodies. One advantage of this approach is that the location of POIs can be ascertained within the tissue architecture (e.g., the basal layer of skin epidermis). This approach also does not require tissue dissociation that may lead to changes in gene expression. One disadvantage is that the tissue fixation or the processing of the tissue for OCT frozen or paraffin sectioning may occlude antibody targets (i.e., antigens). Retrieval of antigens typically requires heat or tissue digestion. Quantification of staining intensities can also be challenging. This is due to variations in staining, section thickness, signal detection, and experimenter bias. Moreover, a limited number of markers can be detected simultaneously in most typical laboratory setups. Yet, newer multiplex staining approaches promise to overcome these limitations; examples are imaging mass cytometry and Tyramide signal amplification6,7.
Flow cytometry is another powerful technology to detect proliferating cells. It allows for multiplex detection of markers in the same cells but requires tissue dissociation for most non-hematopoietic cell types. Analysis of proliferating cells is routinely done by the use of dyes that bind DNA (e.g., Propidium Iodide (PI))8. Flow cytometry also permits a more precise determination of cell cycle phases when coupled with the detection of BrdU incorporation9. Although a powerful approach, BrdU/PI flow cytometry does have its disadvantages. It is unable to resolve the G2/M and G0/G1 phases without the inclusion of phase-specific antibodies. However, the number of antibodies that can be used is limited by cellular autofluorescence, spectral spillover of fluorophore emissions, and the use of compensation controls. This limitation markes it more challenging and laborious to co-detect the expression of cell cycle markers with POIs. A more facile approach is to use mass cytometry10,11. This technology uses metal conjugated antibodies that have a narrower detection spectrum. Once cells are stained with metal-tagged antibodies, they are vaporized, and the metals detected by cytometry time-of-flight (CyTOF) mass spectrometry. Due to these properties, mass cytometry enables the multiplex detection of &#62;40 different markers using existing platforms10,11. In addition, it is possible to barcode samples with metals that result in the savings of precious antibodies while reducing sample-to-sample staining variability. On the other hand, mass cytometry does have several disadvantages. There are a limited number of commercially available metal-tagged antibodies for non-blood derived cells. Quantification of DNA content is less sensitive compared to the use of fluorescent DNA dyes and mass cytometry has a reduced dynamic range of signal detection compared to fluorescence flow cytometry.
The protocol described here was designed to analyze cell cycle dynamics from newly isolated keratinocytes (KCs) from mouse skin and characterize cell cycle specific protein expression in these cells using mass cytometry. This protocol can also be used with cultured cells or adapted to other cell types.

<table border="0" cellpadding="0" cellspacing="0" style="width:1157px;" width="1159">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">12-well plate</td>
			<td style="width:237px;">Cell Treat</td>
			<td style="width:259px;">229512</td>
			<td style="width:368px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Intercalator solution</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201192A</td>
			<td>125 &#181;M - Ir intercalator solution</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Paraformaldehyde</td>
			<td style="width:237px;">Electron Microscopy Sciences</td>
			<td style="width:259px;">30525-89-4</td>
			<td>16 %&#160; PFA</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Strainer cap flow tubes</td>
			<td style="width:237px;">Fisher/Corning</td>
			<td style="width:259px;">352235</td>
			<td>35 &#181;m pore size&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cell sieve</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">22363547</td>
			<td>40 &#956;m pore size&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cell detatchment solution</td>
			<td style="width:237px;">CELLnTEC</td>
			<td style="width:259px;">CnT-ACCUTASE-100</td>
			<td>Accutase</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Iodine Solution</td>
			<td style="width:237px;">ThermoFisher/Purdue</td>
			<td style="width:259px;">67618-151-17</td>
			<td>Betadine 7.5%-iodine surgical scrub</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Barcode permeabilization buffer&#160;</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201060</td>
			<td style="width:368px;">Cell-ID 20-Plex Pd Barcoding Kit</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Barcodes</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201060</td>
			<td style="width:368px;">Cell-ID 20-Plex Pd Barcoding Kit</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">pH strips</td>
			<td style="width:237px;">EMD</td>
			<td style="width:259px;">9590</td>
			<td>colorpHast</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">DMEM</td>
			<td style="width:237px;">Hyclone</td>
			<td style="width:259px;">SH30022.01</td>
			<td style="width:368px;">Dulbecco&#8217;s Modified Eagle Media</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Fine forceps</td>
			<td style="width:237px;">Dumont &#38; Fils</td>
			<td style="width:259px;">0109-5-PO</td>
			<td style="width:368px;">Dumostar #5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Curved precision forceps</td>
			<td style="width:237px;">Dumont &#38; Fils</td>
			<td style="width:259px;">0109-7-PO</td>
			<td style="width:368px;">Dumostar #7</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Calibration Beads</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201078</td>
			<td style="width:368px;">EQ Four Element Calibration Beads</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">HBSS</td>
			<td style="width:237px;">Gibco</td>
			<td style="width:259px;">14175-095</td>
			<td>Hank&#39;s Balanced Salt Solution</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Water</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">SH30538.03</td>
			<td style="width:368px;">Hyclone Molecular Biology grade water</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Iododeoxyuridine</td>
			<td style="width:237px;">&#160;Sigma</td>
			<td style="width:259px;">I7125</td>
			<td>IdU</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cryo vials</td>
			<td style="width:237px;">ThermoFisher</td>
			<td style="width:259px;">366656PK</td>
			<td>internal thread</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cell Staining buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201068</td>
			<td style="width:368px;">Maxpar Cell Staining buffer</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Fix &#38; Perm buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201067</td>
			<td style="width:368px;">Maxpar Fix &#38; Perm buffer</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Fix I buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201065</td>
			<td style="width:368px;">Maxpar Fix I buffer</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Phosphate buffered saline</td>
			<td style="width:237px;">Rockland</td>
			<td style="width:259px;">MB-008</td>
			<td>Metal free 10x PBS</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">isopropanol-freezing container</td>
			<td style="width:237px;">ThermoFisher</td>
			<td style="width:259px;">5100-0001</td>
			<td>Mr.Frosty</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Sodium hydroxide</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">BP359-500</td>
			<td>NaOH</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Petri dish</td>
			<td style="width:237px;">Kord-Valmark</td>
			<td style="width:259px;">2900</td>
			<td>Supplied by Genesee 32-107&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">15 mL conical</td>
			<td style="width:237px;">Olympus/Genesee</td>
			<td style="width:259px;">28-101</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">50 mL conical</td>
			<td style="width:237px;">Olympus/Genesee</td>
			<td style="width:259px;">28-106</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">6-well plate</td>
			<td style="width:237px;">Cell Treat</td>
			<td style="width:259px;">229506</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cisplatin</td>
			<td style="width:237px;">Sigma</td>
			<td style="width:259px;">479306</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Dispase II</td>
			<td style="width:237px;">Sigma/Roche</td>
			<td style="width:259px;">4942078001</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">DMSO</td>
			<td style="width:237px;">Sigma</td>
			<td style="width:259px;">D2650</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">FBS</td>
			<td style="width:237px;">Atlanta Biologicals</td>
			<td style="width:259px;">S11150</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Hydrochloric acid</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">A144-212</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Nuclear Antigen Staining&#160; permeabilization buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201063</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Nuclear Antigen Staining buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201063</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Trypan blue</td>
			<td style="width:237px;">Sigma</td>
			<td style="width:259px;">T8154</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Tuberculin syringe</td>
			<td style="width:237px;">BD</td>
			<td style="width:259px;">309626</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Type IV Collagenase&#160;</td>
			<td style="width:237px;">Worthington Bioscience</td>
			<td style="width:259px;">CLSS-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and staining of mouse skin keratinocytes for cell cycle specific analysis of cellular protein expression by mass cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

Table 1 shows the expected cell yields and viability from adult mouse ear (Figure 1) and neonatal skin under non-pathological conditions. The table also shows representative data of animals from a mixed C57/126 background. It is expected that the skin of other strains would result in similar cell yields and viabilities. The approximate yield is dependent on the surface area of skin and indicates that neonatal skin would be a better choice for...

Watch this Scientific Journal Video about Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry at JoVE.com

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

This protocol is significant for two main reasons. Number one, it allows a user to be able to determine the cell cycle states of skin cells that have been isolated from an animal model. It also allows us to be able to analyze protein expression in the discrete phases of the cell cycle.Compared to previous methods of determining cell proliferation, our method permits a more precise determination of the different phases in the cell cycle, and we can also analyze greater than 40 different markers in the various stages of cell division. This greatly enhances the application and versatility of the method. This protocol may be used in cancer research and in any other type of disease where there's a need to determine cell cycle specific protein expression patterns.In addition, this protocol can be modified to other cell types, and in other model organisms. The first time user should focus on getting the best quality cell preparation possible. This requires the timely processing of experimental skin using fresh reagents, minimal tissue digestion time, and careful disposal of the supernatant after centrifugation to preserve the integrity and quantity of cells.To begin, design a metal-tagged antibody panel using a free online panel design software, as described in the manuscript. Then prepare an IdU stock solution by dissolving IdU powder at 10 milligrams per milliliter in 0.1 normal sodium hydroxide at 60 degrees Celsius. After aliquoting IdU stock solution into microcentrifuge tubes, freeze them at minus 20 degrees Celsius for long-term storage.Immediately before use, adjust the pH of the IdU solution to 7.5 with 12 normal hydrochloric acid, and test with a pH strip on a discard aliquot to ensure the solution is at pH 7.5. To prepare a two x paraformaldehyde fixing solution, combine five milliliters of 10 x PBS and one milliliter of 16%PFA with 44 milliliters of pure molecular-grade distilled water. To prepare 100 millimolar cisplatin stock solution, dissolve 300.5 milligrams of cisplatin powder in 10 milliliters of DMSO.For experiments to be used over one day, prepare a 10 millimolar cisplatin working solution. Weigh mice and then determine a dose at 0.1 milligrams of IdU per gram of body weight. Administer the calculated dose of IDU by an intraperitoneal injection, and wait for two hours before harvesting cells.Use a pair of surgical grade scissors to surgically remove the ear of the euthanized mouse previously injected with IdU at the base of the ear. Place the ear on a dry 100 millimeter Petri dish. Carefully separate the anterior from the posterior skin by using fine forceps to create a pocket in the middle or edge of the cut area, then pull the two skin flaps apart, and proceed with both the anterior and posterior skins.Carefully place the anterior and posterior skins of one ear in one well of a 12-well culture plate, filled with one milliliter of freshly-prepared dispase/collagenase solution, with the derma side of the skin touching the solution. Incubate at 37 degrees Celsius for one hour to digest the skin. Place the digested ear or neonatal skin in a clean Petri dish with the epidermis side touching the surface of the dish.Using forceps, flatten out the skin and gently slide the dermis off the epidermis by working from the center to the edges in a circular pattern. With forceps, grab the epidermis at the edges and gently lift it off by peeling it off the surface of the Petri dish. Carefully place the epidermis on pre-warmed cell detachment solution in one well of a plate.Incubate for five minutes at 37 degrees Celsius or 20 minutes at room temperature. Use sterile forceps to grasp the epidermis and scrub by dragging the epidermis against the bottom of the dish to dissociate cells. Add one milliliter of DMEM containing 1%FBS, and pass through a 40 micrometer cell sieve into a collection tube.Rinse well with an additional two milliliters of DMEM and add to the cell suspension. After centrifuging at 120 times g for five minutes, carefully aspirate the supernatant. Resuspend the cell pellet in one to two milliliters of DMEM containing 1%FBS, and pellet the cells again as previously.To label cells to determine live and dead cells, resuspend one to three million cells in one milliliter of DMEM containing 25 micromolar cisplatin and incubate for one minute. Quench by pipetting with an equal volume of FBS. After centrifuging at 120 times g for five minutes, decant the supernatant into a beaker containing diluted bleach, and invert the tubes to drain the remaining solution onto a paper towel.Resuspend the cell pellet in two milliliters of barium cation-free PBS, and centrifuge them again as previously described. To fix the cells, resuspend the cell pellet in one milliliter of barium cation-free PBS. Vortex the cells under continuous low power and add one milliliter of the two x PFA fixation buffer drop-wise.Place on a rocking platform and incubate at room temperature for 10 minutes. After centrifuging at 500 times g for five minutes, carefully decant the supernatant. Wash the cells with two milliliters of barium cation-free PBS, centrifuge again under the same conditions, and repeat the washing step.Finally, resuspend the pellet in two milliliters of barium cation-free PBS. Centrifuge the cells at 500 times g for five minutes and carefully decant the supernatant. Resuspend the cells in one milliliter of one x fixation buffer, and incubate at room temperature for 10 minutes and repeat the centrifugation.To wash the cells, add one milliliter of barcode permeabilization buffer. During cell centrifugation at 500 times g for five minutes, prepare barcodes by adding 100 microliters of barcode permeabilization buffer to them, and mix immediately. Resuspend the cell pellet in 800 microliters of barcode permeabilization buffer.Add barcode solution to the cells, mix, and incubate at room temperature for 30 minutes. Centrifuge at 500 times g for five minutes. Wash cells in two milliliters of cell staining buffer and centrifuge again.Next, resuspend one to three million cells in one milliliter of nuclear antigen staining buffer working solution, and incubate at room temperature for 30 minutes. After centrifuging at 500 times g for five minutes, carefully decant the supernatant. Resuspend the cells in one milliliter of nuclear antigen staining permeabilization buffer and centrifuge again.After repeat resuspension and centrifugation, resuspend the cell pellet in the residual volume with gentle vortexing. Add 50 microliters of intracellular antibody cocktail, mix, and incubate at room temperature for 45 minutes. Add two milliliters of cell staining buffer.Centrifuge again under the same conditions as in the previous step, and remove the supernatant. Resuspend the pellet in two milliliters of cell staining buffer, centrifuge at 500 times g for five minutes, and repeat the resuspending and centrifugation. Resuspend the cells in one milliliter of intercalation solution, and store for one to three days at four degrees Celsius.After centrifuging the cells again, wash the pellet with two milliliters of cell staining buffer, centrifuge under the same conditions, and perform two additional washes with two milliliters of water, with the same centrifugation. Resuspend the cell pellet at a concentration of one million cells per milliliter with diluted EQ bead solution, and then run the samples on the mass cytometer. Expected cell yields and viability from adult mouse ear and neonatal skin are determined.The approximate yield depends on the surface area of the skin. Neonatal skin is a better choice for experiments that require larger numbers of cells. The basic gating strategy to select for intact, single, and viable cells, after normalization and deconvolution of barcoded mass cytometry data, is shown.The percentage of viable cells may be indicative of the quality or condition of the cells prior to staining. Differences in viability between cultured carcinoma cells versus isolated mouse ear cells are shown, too. Viable cells were gated on event length versus CD45 to exclude hematopoietic cells.The inclusion of lineage markers allows for the selection of cell populations of interest in different cell cycle phases. The basic cell cycle profile is obtained by the detection of BrdU versus PI in fluorescent-based flow cytometry. However, inclusion of other proliferation markers with mass cytometry, allows a more precise identification of cell cycle phases.Following this approach, cell cycle profiles can be constructed for different cell types or experimental conditions, such as the profiles for CD45 negative versus CD45 positive cells from the same experiment. In another example, analysis of markers that define the EGFR and mTOR PI3K signaling pathways in the G-one phase, revealed similar expression profiles in CD45 negative cells, shown in orange, and CD45 positive cells, shown in blue. Mass cytometry requires a lot of high-quality cells.If a user is interested in a rare cell population, higher numbers of cells may be needed. Isolated live cells can be used for DNA or RNA analysis, biochemical studies, or can be used in tissue culture prior to the fixation and staining for mass cytometry. The user should use personal protection equipment and a safety cabinet when necessary, when working with paraformaldehyde, hydrochloric acid, or sodium hydroxide.

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JoVE Journal

Developmental Biology

7.3K Views.  University of Colorado Anschutz Medical Campus. This protocol is significant for two main reasons. Number one, it allows a user to be able to determine the cell cycle states of skin cells that have been isolated from an animal model. It also allows us to be able to analyze protein expression in the discrete phases of the cell cycle.Compared to previous methods of determining cell proliferation, our method permits a more precise determination of the different phases in the cell cycle, and we can also analyze greater than 40 different...