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 mark....

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 o....

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