Name: isolation and staining of mouse skin keratinocytes for cell cycle specific analysis of cellular protein expression by mass cytometry
Uploaded: 2019-05-09T13:30:00
Description: 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

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 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.

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	<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 ...

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