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.

<table><tbody><tr><td>12-well plate</td><td>Cell Treat</td><td>229512</td><td></td></tr><tr><td>Intercalator solution</td><td>Fluidigm</td><td>201192A</td><td>125 &amp;micro;M - Ir intercalator solution</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>30525-89-4</td><td>16 %&amp;nbsp; PFA</td></tr><tr><td>Strainer cap flow tubes</td><td>Fisher/Corning</td><td>352235</td><td>35 &amp;micro;m pore size&amp;nbsp;</td></tr><tr><td>Cell sieve</td><td>Fisher</td><td>22363547</td><td>40 &amp;mu;m pore size&amp;nbsp;</td></tr><tr><td>Cell detatchment solution</td><td>CELLnTEC</td><td>CnT-ACCUTASE-100</td><td>Accutase</td></tr><tr><td>Iodine Solution</td><td>ThermoFisher/Purdue</td><td>67618-151-17</td><td>Betadine 7.5%-iodine surgical scrub</td></tr><tr><td>Barcode permeabilization buffer&amp;nbsp;</td><td>Fluidigm</td><td>201060</td><td>Cell-ID 20-Plex Pd Barcoding Kit</td></tr><tr><td>Barcodes</td><td>Fluidigm</td><td>201060</td><td>Cell-ID 20-Plex Pd Barcoding Kit</td></tr><tr><td>pH strips</td><td>EMD</td><td>9590</td><td>colorpHast</td></tr><tr><td>DMEM</td><td>Hyclone</td><td>SH30022.01</td><td>Dulbecco&amp;rsquo;s Modified Eagle Media</td></tr><tr><td>Fine forceps</td><td>Dumont &amp;amp; Fils</td><td>0109-5-PO</td><td>Dumostar #5</td></tr><tr><td>Curved precision forceps</td><td>Dumont &amp;amp; Fils</td><td>0109-7-PO</td><td>Dumostar #7</td></tr><tr><td>Calibration Beads</td><td>Fluidigm</td><td>201078</td><td>EQ Four Element Calibration Beads</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175-095</td><td>Hank&#039;s Balanced Salt Solution</td></tr><tr><td>Water</td><td>Fisher</td><td>SH30538.03</td><td>Hyclone Molecular Biology grade water</td></tr><tr><td>Iododeoxyuridine</td><td>&amp;nbsp;Sigma</td><td>I7125</td><td>IdU</td></tr><tr><td>Cryo vials</td><td>ThermoFisher</td><td>366656PK</td><td>internal thread</td></tr><tr><td>Cell Staining buffer</td><td>Fluidigm</td><td>201068</td><td>Maxpar Cell Staining buffer</td></tr><tr><td>Fix &amp;amp; Perm buffer</td><td>Fluidigm</td><td>201067</td><td>Maxpar Fix &amp;amp; Perm buffer</td></tr><tr><td>Fix I buffer</td><td>Fluidigm</td><td>201065</td><td>Maxpar Fix I buffer</td></tr><tr><td>Phosphate buffered saline</td><td>Rockland</td><td>MB-008</td><td>Metal free 10x PBS</td></tr><tr><td>isopropanol-freezing container</td><td>ThermoFisher</td><td>5100-0001</td><td>Mr.Frosty</td></tr><tr><td>Sodium hydroxide</td><td>Fisher</td><td>BP359-500</td><td>NaOH</td></tr><tr><td>Petri dish</td><td>Kord-Valmark</td><td>2900</td><td>Supplied by Genesee 32-107&amp;nbsp;</td></tr><tr><td>15 mL conical</td><td>Olympus/Genesee</td><td>28-101</td><td></td></tr><tr><td>50 mL conical</td><td>Olympus/Genesee</td><td>28-106</td><td></td></tr><tr><td>6-well plate</td><td>Cell Treat</td><td>229506</td><td></td></tr><tr><td>Cisplatin</td><td>Sigma</td><td>479306</td><td></td></tr><tr><td>Dispase II</td><td>Sigma/Roche</td><td>4942078001</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher</td><td>A144-212</td><td></td></tr><tr><td>Nuclear Antigen Staining&amp;nbsp; permeabilization buffer</td><td>Fluidigm</td><td>201063</td><td></td></tr><tr><td>Nuclear Antigen Staining buffer</td><td>Fluidigm</td><td>201063</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Tuberculin syringe</td><td>BD</td><td>309626</td><td></td></tr><tr><td>Type IV Collagenase&amp;nbsp;</td><td>Worthington Bioscience</td><td>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 ...

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

Research

JoVE Journal

Developmental Biology

7.4K Views.  University of Colorado Anschutz Medical Campus. 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.

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

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.

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 ...

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER+ and R26-SmoM2YFP+ mice in this protocol); 2) inducing SmoM2YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

Medicine

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Immunology and Infection

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Preparing Mouse Thymic Epithelial Cells for Analysis of Intracellular Molecules

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the thymic cortex induce early T cell development and positive selection of cortical thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla attract positively selected thymocytes from the cortex and establish self-tolerance in T cells. A variety of molecules, including DLL4 and beta5t expressed in cTECs, as well as Aire and CCL21 expressed in mTECs, contribute to thymus function supporting T cell development and selection. Flow cytometric analysis of functionally relevant molecules in cTECs and mTECs is useful to improve our understanding of the biology of TECs, even though current methods for the preparation of single-cell suspensions of TECs can retrieve only a small fraction of TECs (approximately 1% for cTECs and approximately 10% for mTECs) from young adult mouse thymus. Because many of these functionally relevant molecules in TECs are localized within the cells, we describe our protocols for the preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis.

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric AnalysisMechanisms

We describe protocols for the preparation of single-cell suspension of mouse thymic epithelial cells and the staining of intracellular molecules for flow cytometric analysis.

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Biology

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...