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 150Nd (Neodymium)-pRETINOBLASTOMA proteinSer807/811 (pRB)13. Add additional metal-tagged antibodies that do not overlap in their channel signal14.</li>
	<li>Prepare an IdU stock solution. Dissolve IdU powder at 10 mg/mL in 0.1 N NaOH at 60 &#176;C. Aliquot IdU stock solution into microcentrifuge tubes and freeze at -20 &#176;C for long-term storage.
	<ol>
		<li>Adjust the pH of IdU solution to 7.5 with 12 N HCl immediately before use. Test with a pH strip on a discard aliquot to ensure the solution is at pH 7.5. 
		NOTE: Prolonged time (&#62;5 min) of IdU at pH 7.5 will result in precipitation and a fresh aliquot of IdU is needed when this happens.</li>
		<li>Use appropriate personal protective equipment (e.g., gloves, lab coat, and safety glasses) and work in a safety cabinet when handling NaOH or HCl solutions.</li>
	</ol>
	</li>
	<li>Prepare a 2x paraformaldehyde (PFA) fixing solution. Combine 5 mL of 10x PBS (pH 7.5) and 1 mL of 16% PFA with 44 mL of pure molecular grade dH2O (3.2% final concentration). 
	NOTE: A stock of PFA can be prepared as previously published15 or purchase 16% stocks free of contaminating metals.
	<ol>
		<li>Use appropriate personal protective equipment and work in a safety cabinet when handling PFA powder and solutions.</li>
	</ol>
	</li>
	<li>Prepare barium (Ba2+) free 1x PBS by combining 5 mL of metal-free 10x PBS (pH 7.5) with 45 mL of pure molecular grade dH2O. 
	NOTE: The quality of PBS and other reagents is very important to avoid contaminating metals that can introduce background noise during analysis of mass cytometric data.</li>
	<li>Prepare a 100 mM cisplatin stock solution. Dissolve 300.5 mg of cisplatin powder in 10 mL of DMSO. Store in aliquots at -80 &#176;C. Prepare a 10 mM cisplatin working solution for experiments to be used over 1 d.</li>
	<li>Prepare epidermis/dermis separation solutions. Prepare a 20x dispase stock solution by dissolving 30 mg in 1 mL of HBSS or PBS. Filter sterilize through a 0.22 &#181;m filter and store in aliquots at -20 &#176;C. Prepare a 1x type IV collagenase stock solution at 1 mg/mL in HBSS, filter sterilize through a 0.22 &#181;m filter, and store aliquots at -20 &#176;C.</li>
</ol>
2. Labeling of S phase by IdU incorporation
<ol>
	<li>Weigh mice. Use P1-P3 neonatal pups or 8-10 weeks old adult mice and use male or female mice from a C57BL/6, FvB, or 129 backgrounds. Determine a dose at 0.1 mg IdU (pH 7.5)/g body weight. For example, a 25 g mouse requires 0.25 mL of IdU.</li>
	<li>Administer the dose of IdU by an intraperitoneal injection and wait for 2 h before harvesting cells. Use a tuberculin syringe to inject neonatal pups. 
	NOTE: A dose of 2 mg/mL of IdU can be used with tissue culture cells with a 1 h incubation at 37 &#176;C before harvesting and labeling for mass cytometry.</li>
</ol>
3. Isolation of cells for labeling
<ol>
	<li>Thaw dispase and collagenase stock solutions. Dilute the 20x dispase stock solution to 1x (1.5 mg/mL) in sterile HBSS or PBS. Keep on ice until ready to use.</li>
	<li>Combine 2 volumes of dispase with 1 volume of collagenase in a total volume that allows the skin to float freely. For example, digestion of 5 neonatal mouse skins can be floated on 8 mL of 1x dispase with 4 mL of collagenase in a 100 mm Petri dish.</li>
	<li>Follow approved methods to euthanize experimental mice (e.g., isoflurane overdose/toe pinch check or CO2 inhalation/cervical dislocation). Clean the adult ear skin with an iodine solution (see Table of Materials) and rinse with sterile water when isolated cells are to be used for tissue culture.</li>
	<li>Surgically remove the ear at the base (Figure 1A). Rinse ears in sterile PBS when isolated cells is to be used for tissue culture. Place on a dry 100 mm Petri dish.</li>
	<li>Separate carefully anterior from posterior skin by initially creating a pocket in the middle or edge of the cut area using fine forceps and pulling the two skin flaps apart ( Figure 1B-D). Proceed with both the anterior and posterior skins. 
	NOTE: Detailed instructions to dissect neonatal mouse skin are provided by Litchi et al.16 In addition, the use of a dissecting scope may assist in the separation of anterior from posterior skin and identification of epidermis/dermal sides of dissected skin: hair is visible on the epidermis side whereas the dermis will have a gelatinous appearance.</li>
	<li>Carefully place the anterior and posterior skins of the ear with the dermis side of the skin touching the dispase/collagenase solution ( Figure 1E). Use 1 mL for both the anterior and posterior skin of a single ear per well of a 12 well culture plate. Incubate at 37 &#176;C for 1 h. Alternatively, float skins on 1x dispase solution at 4 &#176;C for 16-18 h.3 
	NOTE: Work in a tissue culture hood with sterile technique when cells are to be cultured.</li>
	<li>Place the digested skin with the epidermis side touching the surface of a clean Petri dish. Flatten out the skin and gently slide the dermis off the epidermis by working from center to the edges in a circular pattern.</li>
	<li>Discard the dermis or digest it further to liberate fibroblasts for tissue culture or analysis16. 
	NOTE: The dermis will be darker in appearance and have a gelatinous and sticky composition. The dermis should easily come off. Failure or difficulty in removing the dermis suggests insufficient tissue digestion. However, extended incubation in digestion buffer will reduce cell viability. The epidermis will remain on the Petri dish and will have a bleached appearance.</li>
	<li>Gently lift off the epidermis by grabbing at the edges and peel it off the surface of the Petri dish. Carefully place the epidermis on pre-warmed cell detachment solution (see Table of Materials) for 5 min at 37 &#176;C or 20 min at RT. Use 500 &#181;L of cell detachment solution for 2 adult ear epidermises per well of a 12 well culture plate and use 750 &#181;L of cell detachment solution for a single neonatal epidermis per well of a 6 well culture plate.</li>
	<li>Grasp the epidermis using sterile forceps and scrub by dragging the epidermis against the bottom of the dish to dissociate cells. Add 1 mL of DMEM containing 1% FBS (0.01 mL) and pass through a 40 &#956;m cell sieve into a collection tube. Rinse well with an additional 2 mL of DMEM and add to the cell suspension.</li>
	<li>Centrifuge at 120 x g for 5 min. Aspirate the supernatant carefully.</li>
	<li>Resuspend cell pellet in 1-2 mL of DMEM containing 1% FBS. Determine the cell and % live cell counts using Trypan Blue and a hemocytometer or automated counting device. Pellet cells as described in step 3.11. 
	NOTE: Cells can be cultured in appropriate tissue culture media after Step 3.12.17</li>
</ol>
4. Cisplatin labeling to determine live/dead cells
<ol>
	<li>Resuspend 1-3 x 106 cells per 1 mL of DMEM containing 25 &#181;M cisplatin (2.5 &#181;L of stock/mL). Incubate for 1 min and quench by pipetting with an equal volume of FBS (e.g., 1 mL).</li>
	<li>Centrifuge at 120 x g for 5 min. Decant supernatant into a beaker containing diluted bleach and invert tubes to drain remaining solution onto a paper towel. Tap gently to liberate solution remaining on pellet and sidewall of the tube.</li>
	<li>Resuspend cell pellet in 2 mL of Ba+2-free PBS. Pellet cells as described in Step 4.2. 
	NOTE: It is recommended to fix cells if they cannot be stained immediately.</li>
</ol>
5. Fixation of cisplatin labeled cells (optional)
<ol>
	<li>Resuspend 1-3 x 106 cisplatin labeled cells in 1 mL of Ba+2-free PBS. Vortex cells under continuous low power and add dropwise 1 mL (1 volume) of the 2x PFA fixation buffer. Incubate at RT for 10 min on a rocking platform.</li>
	<li>Pellet cells as described in step 4.2, but centrifuge at 500 x g for 5 min.</li>
	<li>Wash cells with 2 mL of Ba+2-free PBS and pellet cells as described in Step 5.2. Repeat Step 5.3 once.</li>
	<li>Resuspend the pellet in 2 mL of Ba+2-free PBS. Store at 4&#176;C for &#60;3 d. Add FBS to 3% (e.g., 0.3 mL of FBS/mL of cells) of the total volume if storing longer &#62;3 d, then mix and freeze at -80 &#176;C. 
	NOTE: Fixation may affect the detection of certain epitopes. The effects of fixation on antibody signal&#160;need to be determined empirically.</li>
</ol>
6. Barcoding of samples (optional but recommended)
<ol>
	<li>Pellet cells as described in Step 5.2 and then resuspend 1-3 x 106 cells in 1 mL of 1x Fixation buffer (see Table of Materials). Incubate at RT for 10 min. Pellet cells as described in Step 5.2.</li>
	<li>Wash cells with 1 mL of Barcode permeabilization buffer (see Table of Materials). Pellet cells as described in Step 5.2. Repeat Step 6.2 once.</li>
	<li>Add 100 &#181;L of Barcode permeabilization buffer to barcodes (see Table of Materials)18 and mix immediately while the cells from Step 6.2 are pelleting.</li>
	<li>Resuspend the cell pellet in 800 &#181;L of Barcode permeabilization buffer. Add barcode solution to cells, mix and incubate at RT for 30 min. Pellet cells as described in Step 5.2. Wash cells in 2 mL of Cell staining buffer (see Table of Materials) and pellet again. 
	NOTE: Up to 20 samples can be labeled with various combinations of Palladium metals18. Other barcoding strategies are described elsewhere15.</li>
</ol>
7. Labeling of cells for mass cytometry
<ol>
	<li>Resuspend 1-3 x 106 cells in 1 mL of Nuclear Antigen Staining buffer working solution (see Table of Materials). Combine samples into a single tube for subsequent steps when using barcoded samples. Incubate at RT for 30 min. Pellet as described in Step 5.2.</li>
	<li>Resuspend 1-3 x 106 cells in 2 mL of Nuclear Antigen Staining permeabilization buffer (see Table of Materials). Scale as necessary. For example, use 6 mL of buffer for 10 combined samples with a cell count of 9 x 106 cells. Pellet as described in Step 5.2.</li>
	<li>Repeat Step 7.2. Gently&#160;vortex the cell pellet in the residual volume left in the tube.</li>
	<li>Add 50 &#181;L of intracellular antibody cocktail per 1-3 x 106 cells. Scale as necessary. For example, use 150 &#956;L of the antibody cocktail for 10 combined samples with a cell count of 9 x 106 cells. Mix and incubate at RT for 45 min. Add 2 mL of Cell Staining buffer and pellet as described in step 5.2.</li>
	<li>Resuspend 1-3 x 106 cells pellet in 2 mL of Cell Staining buffer. Pellet cells as described in Step 5.2. Repeat Step 7.5 once. 
	NOTE: Other buffer solutions can be used for staining extracellular membrane or cytoplasmic markers and experimenter may have to optimize staining if there is a need to detect epitopes in different cellular locations. Cells can also be fixed in PFA after Step 7.5 when using live cells for staining.</li>
	<li>Resuspend 1-3 x 106 cells in 1 mL of intercalation solution and store for 1-3 d at 4 &#730;C.
	<ol>
		<li>Store cells at -80 &#176;C in DMSO containing the solution for &#62;3 d. Pellet cells as described in Step 5.2 if cells are in intercalation solution. Resuspend pellet (1-3 x 106 cells) in 1 mL of Cell staining buffer and pellet cells as described in Step 5.2. Resuspend pellet in 1 mL of 10% DMSO/90% FBS19, transfer to a cryovial, place in an isopropanol-freezing container, and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Pellet cells as described in Step 5.2. Wash 1-3 x 106 cells with 2 mL of Cell Staining buffer, pelleting as described in Step 5.2. Perform two additional washes with 2 mL water, pelleting as described in Step 5.2.</li>
	<li>Dilute EQ calibration beads 1:9 in water (stock solution at 3.3 x 105 beads/mL).</li>
	<li>Resuspend the cell pellet at a concentration of 1 x 106 cells/mL with diluted EQ bead solution. Filter cells through 35 &#181;m strainer cap flow tubes.</li>
	<li>Run samples on the mass cytometer and acquire data. The data will be deposited in a Flow Cytometry Standard (FCS) file format.</li>
</ol>
8. Processing and analysis of mass cytometry data files
<ol>
	<li>Normalize FCS files. Use a free program available at &#160;https://github.com/nolanlab/bead-normalization/releases/latest20</li>
	<li>Deconvolute barcoded FCS files. Separate out the pooled barcode population into separate barcoded files with a free program available at &#160;https://github.com/nolanlab/single-cell-debarcoder/releases/latest18</li>
	<li>Analyze normalized FCS files. Use commercial21,22 or freeware programs (see web.stanford.edu/group/nolan/resources.html).</li>
</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.

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			<td>40 &#956;m pore size&#160;</td>
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			<td>Betadine 7.5%-iodine surgical scrub</td>
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			<td height="70" style="height:70px;width:293px;">Barcode permeabilization buffer&#160;</td>
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			<td style="width:259px;">201060</td>
			<td style="width:368px;">Cell-ID 20-Plex Pd Barcoding Kit</td>
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			<td height="35" style="height:35px;width:293px;">Iododeoxyuridine</td>
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			<td style="width:259px;">366656PK</td>
			<td>internal thread</td>
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			<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>
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			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201065</td>
			<td style="width:368px;">Maxpar Fix I buffer</td>
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			<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>
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			<td height="70" style="height:70px;width:293px;">isopropanol-freezing container</td>
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			<td style="width:259px;">5100-0001</td>
			<td>Mr.Frosty</td>
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			<td style="width:237px;">Kord-Valmark</td>
			<td style="width:259px;">2900</td>
			<td>Supplied by Genesee 32-107&#160;</td>
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			<td height="35" style="height:35px;width:293px;">15 mL conical</td>
			<td style="width:237px;">Olympus/Genesee</td>
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			<td height="35" style="height:35px;width:293px;">50 mL conical</td>
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			<td height="35" style="height:35px;width:293px;">6-well plate</td>
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			<td height="35" style="height:35px;width:293px;">Cisplatin</td>
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			<td height="35" style="height:35px;width:293px;">Dispase II</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:293px;">FBS</td>
			<td style="width:237px;">Atlanta Biologicals</td>
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			<td height="35" style="height:35px;width:293px;">Hydrochloric acid</td>
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			<td style="width:259px;">A144-212</td>
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			<td height="70" style="height:70px;width:293px;">Nuclear Antigen Staining&#160; permeabilization buffer</td>
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			<td style="width:259px;">201063</td>
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			<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>
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			<td height="35" style="height:35px;width:293px;">Trypan blue</td>
			<td style="width:237px;">Sigma</td>
			<td style="width:259px;">T8154</td>
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			<td height="70" style="height:70px;width:293px;">Tuberculin syringe</td>
			<td style="width:237px;">BD</td>
			<td style="width:259px;">309626</td>
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		<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

Isolierung und Färbung der Maushaut Keratinozyten für Zellzyklus spezifische Analyse der zellulären Proteinexpression durch Massenzytometrie

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 ...

isolation-staining-mouse-skin-keratinocytes-for-cell-cycle-specific

Research

JoVE Journal

Developmental Biology

7.4K Aufrufe.  University of Colorado Anschutz Medical Campus. Dieses Protokoll ist aus zwei Hauptgründen von Bedeutung. Nummer eins, ermöglicht es einem Benutzer, die Zellzykluszustände von Hautzellen zu bestimmen, die aus einem Tiermodell isoliert wurden. Es ermöglicht uns auch, die Proteinexpression in den diskreten Phasen des Zellzyklus zu analysieren.Im Vergleich zu früheren Methoden zur Bestimmung der Zellproliferation ermöglicht unsere Methode eine genauere Bestimmung der verschiedenen Phasen im Zellzyklus, und wir können auch mehr a...

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

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D Organotypische Cokulturmodell Unterstützung Mark Thymusepithel Zellproliferation, Differenzierung und Genexpression Promiscuous

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Forschung

Entwicklungsbiologie

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Herstellung von genetisch veränderten Organotypische Hautkulturen Mit devitalisierten Menschen Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Schritt spezifische Sortierung der Maus Spermatiden durch Durchflusszytometrie

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Quantitative Analyse der Proteinexpression zu studieren Lineage Spezifikation in Maus Präimplantationsembryonen

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

Isolieren von Haarfollikelstammzellen und epidermalen Keratinozyten von Dorsal Mäusehaut

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Identifizierung von Erythromyeloid Progenitoren und deren Nachkommen in der Maus Embryo Durch Durchflusszytometrie

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

Generation und Kultivierung der primären menschlichen Keratinozyten von Erwachsenen

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Eine vereinfachte und effiziente Methode zur primären menschlichen Keratinozyten aus Erwachsenen Hautgewebe zu isolieren

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

Hämogene Reprogrammierung menschlicher Fibroblasten durch erzwungene Expression von Transkriptionsfaktoren

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.