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

Summary

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.

Abstract

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.

Introduction

The skin is one of the largest organs of the body and its large surface is continuously exposed to the external environment. Therefore the skin has to protect the organism from potential threats in order to maintain homeostasis, both by physical means, and by providing active protection toward potential pathogen entry. In a similar way to the gut and lung mucosa, the skin homes a variety of immune cells that continuously interact with the epithelium in order to maintain immune surveillance. This complex system that involves both immune and non-immune cells of the skin has been acknowledged since the early days of immunology, when in 1978 the term "Skin-associated lym....

Protocol

Study approval: The experimental protocols were approved by the Italian Ministry of Health (Rome, Italy) according to the Decreto legislativo 27 gennaio 1992, n. 116 "Attuazione della Direttiva n. 86/609/CEE in materia di protezione degli animali utilizzati a fini sperimentali o ad altri fini scientifici."

1. Obtaining Skin Samples

1. Euthanize the mouse by cervical dislocation.
2. Ear:
   1. Cut out the hairless part of the ears of the mouse.
   2. Separate dorsal and ventral side by pulling them apart with forceps. Remove any remaining cartilages by gently scraping the inner part of the ears with forceps....

Results

Skin from different region of the body was digested according to the protocol described and stained with the indicated antibodies. The gating strategy is depicted in Figure 1. First select live cells (DAPI- cells), then gate on singlets (FSC-A vs. FSC-W) and on cells with lymphocyte morphology (FSC-A vs. SSC-A). When indicated, CD45⁺ cells from hematopoietic origin are selected.

DCs are labeled with a.......

Discussion

We have described a method for the preparation of single-cell suspensions from different mouse skin regions. The method of digestion we have adopted, not only gives high yields but also preserves the expression of surface markers, which is fundamental for the subsequent FACS analysis.

The use of a cocktail of collagenase I and II and thermolysin, guarantees minimum batch to batch differences in enzyme activity making this method highly reproducible. Other published methods rely on different en.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Supplemented RPMI 1640 medium			RPMI1640 supplemented w/ L-glu, pen-strep w/o beta mercapto ethanol
PBS
FBS
Liberase TM (Enzyme Mix)	Roche	5401119001	resuspend [2.5 mg/ml] in dH2O; store aliquots at -20 °C
Dnase I	Sigma	D4263-1VL	resuspend in RPMI 1640 w/o serum [1,000 U/ml]
Materials
Surgical Scissors
Surgical Forceps
Surgical Scalpel
35 mm Petri dishes
66 mm Petri dishes
100 mm Petri dishes
70 µm Cell Strainers	BD	352350
Digestion cocktail			Dilute in RPMI 5% FBS
LIberase TM			300 µg/ml
DNAse1			50 U/ml
Antibodies
Name	Company	Clone	Label
aCD45.2		104	PE-Cy5.5
aCD11c		N418	PE-Cy7
CD11b		M1/70	FITC
F4/80		A 3-1	APC-Cy7
aCD4		Gk1.5	APC-Cy7
aCD8		53-6.7	PE
aCD64		X54-5/7.1	PE-Cy7
aCD207		4C7	APC