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

Summary

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.

Abstract

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 >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.

Introduction

Correlation of 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 tissues^1,2,3. 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 amplification^6,7.

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 cytometry^10,11. 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 >40 different markers using existing platforms^10,11. 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.

Protocol

The University of Colorado Anschutz Medical Campus' Institutional Animal Care and Use Committee approved the animal experiments described in this protocol.

1. Preparations

1. 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 (p)-HISTONEH3^Ser28 (pHH3), and ¹⁵⁰Nd (Neodymium)-pRETINOBLASTOMA protein^Ser807/811 (pRB)¹³. Add additional metal-tagged antibodies that do not overlap in their channel signal¹⁴.
2. Prepare an IdU stock solution. Dissolve IdU powder at 10 mg/mL in 0.1 N NaOH at 60 °C. Aliquot IdU stock solution into microcentrifuge tubes and freeze at -20 °C for long-term storage.
   1. 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 (>5 min) of IdU at pH 7.5 will result in precipitation and a fresh aliquot of IdU is needed when this happens.
   2. 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.
3. 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.
   1. Use appropriate personal protective equipment and work in a safety cabinet when handling PFA powder and solutions.
4. 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.
5. Prepare a 100 mM cisplatin stock solution. Dissolve 300.5 mg of cisplatin powder in 10 mL of DMSO. Store in aliquots at -80 °C. Prepare a 10 mM cisplatin working solution for experiments to be used over 1 d.
6. 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 µm filter and store in aliquots at -20 °C. Prepare a 1x type IV collagenase stock solution at 1 mg/mL in HBSS, filter sterilize through a 0.22 µm filter, and store aliquots at -20 °C.

2. Labeling of S phase by IdU incorporation

1. 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.
2. 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 °C before harvesting and labeling for mass cytometry.

3. Isolation of cells for labeling

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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 °C for 1 h. Alternatively, float skins on 1x dispase solution at 4 °C for 16-18 h.³
   NOTE: Work in a tissue culture hood with sterile technique when cells are to be cultured.
7. 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.
8. 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.
9. 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 °C or 20 min at RT. Use 500 µL of cell detachment solution for 2 adult ear epidermises per well of a 12 well culture plate and use 750 µL of cell detachment solution for a single neonatal epidermis per well of a 6 well culture plate.
10. 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 μm cell sieve into a collection tube. Rinse well with an additional 2 mL of DMEM and add to the cell suspension.
11. Centrifuge at 120 x g for 5 min. Aspirate the supernatant carefully.
12. 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

4. Cisplatin labeling to determine live/dead cells

1. Resuspend 1-3 x 10⁶ cells per 1 mL of DMEM containing 25 µM cisplatin (2.5 µL of stock/mL). Incubate for 1 min and quench by pipetting with an equal volume of FBS (e.g., 1 mL).
2. 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.
3. 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.

5. Fixation of cisplatin labeled cells (optional)

1. 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.
2. Pellet cells as described in step 4.2, but centrifuge at 500 x g for 5 min.
3. Wash cells with 2 mL of Ba⁺²-free PBS and pellet cells as described in Step 5.2. Repeat Step 5.3 once.
4. Resuspend the pellet in 2 mL of Ba⁺²-free PBS. Store at 4°C for <3 d. Add FBS to 3% (e.g., 0.3 mL of FBS/mL of cells) of the total volume if storing longer >3 d, then mix and freeze at -80 °C.
   NOTE: Fixation may affect the detection of certain epitopes. The effects of fixation on antibody signal need to be determined empirically.

6. Barcoding of samples (optional but recommended)

1. 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.
2. 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.
3. Add 100 µL of Barcode permeabilization buffer to barcodes (see Table of Materials)¹⁸ and mix immediately while the cells from Step 6.2 are pelleting.
4. Resuspend the cell pellet in 800 µL 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.

7. Labeling of cells for mass cytometry

1. 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.
2. 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.
3. Repeat Step 7.2. Gently vortex the cell pellet in the residual volume left in the tube.
4. Add 50 µL of intracellular antibody cocktail per 1-3 x 10⁶ cells. Scale as necessary. For example, use 150 μL 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.
5. 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.
6. Resuspend 1-3 x 10⁶ cells in 1 mL of intercalation solution and store for 1-3 d at 4 ˚C.
   1. Store cells at -80 °C in DMSO containing the solution for >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 °C.
7. 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.
8. Dilute EQ calibration beads 1:9 in water (stock solution at 3.3 x 10⁵ beads/mL).
9. Resuspend the cell pellet at a concentration of 1 x 10⁶ cells/mL with diluted EQ bead solution. Filter cells through 35 µm strainer cap flow tubes.
10. Run samples on the mass cytometer and acquire data. The data will be deposited in a Flow Cytometry Standard (FCS) file format.

8. Processing and analysis of mass cytometry data files

1. Normalize FCS files. Use a free program available at  https://github.com/nolanlab/bead-normalization/releases/latest²⁰
2. Deconvolute barcoded FCS files. Separate out the pooled barcode population into separate barcoded files with a free program available at  https://github.com/nolanlab/single-cell-debarcoder/releases/latest¹⁸
3. Analyze normalized FCS files. Use commercial^21,22 or freeware programs (see web.stanford.edu/group/nolan/resources.html).

Results

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...

Discussion

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 skin^16,25. These studies also include protocols for the isolation of KCs for flow cytometry²⁶. However, a detailed protocol has not been previously described t...

Materials

List of materials used in this article

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