Summary

다른 마우스 피부 지역에서 Cytofluorimetric 분석을위한 단일 세포 현탁액의 준비

Published: April 20, 2016
doi:

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 lymphoid tissue” (SALT) 1 was first coined to describe such complexity.

The large number of immune cells residing in the skin helps the organism not only to fight potential invasions, but also to orchestrate wound healing and to maintain tolerance toward self-antigens and the skin microbiota 2,3.

Amongst skin resident immune cells, Dendritic Cells (DCs) have a crucial role in shaping immune responses and maintaining homeostasis 4. Dermal DCs and Langerhans cells readily respond to pathogen invasions and, upon migration to lymph nodes activate T cells and induce the expression of skin homing receptors on the newly generated effector T cells 5. DCs also have a fundamental role in regulating skin homeostasis. DCs migrating from the dermis to the lymph nodes in homeostatic conditions transport skin sequestered antigens with the purpose of inducing T cell tolerance mostly through the differentiation of regulatory T cells (Tregs) specific for skin antigens 6-8.

T cells represent the most abundant population of immune cells in the skin. Not only do they infiltrate the skin during an infection but they also represent a stable population among skin resident lymphocytes 9,10. Both CD8+ and CD4+ resident memory (rm)T cells reside in the skin and, following an infection, can respond long before effector T cells are recruited from the blood. In the skin, also resides a population of tissue resident Tregs capable of maintaining tolerance toward skin antigens and tissue homeostasis. These cells are rapidly and potently activated after exposure to their cognate antigen and have therefore been defined as memory Tregs 11,12.

Along with DCs and T cells, many innate immune cells, such as NK cells, gamma delta T cells, group 2 ILCs 13, Mast Cells and Macrophages, populate the skin and contribute to host protection. To analyze such a complex environment, it is mandatory to obtain single-cell suspensions from skin specimens with high efficiency while at the same time preserving the expression of surface markers for flow cytofluorimetric analysis or sorting.

Murine skin presents an outer epidermal layer, constituted mainly of keratinocytes, Langerhans cells and dendritic epidermal T cells; and the dermal layer beneath. The dermis homes most of the immune cells and is made by an extracellular matrix in which collagen fibers are the most abundant, especially collagen-I and collagen-IV. Unlike human skin, mouse skin is covered in fur, and thus more populated with hair follicles. It is, also, much thinner than human skin and contains a muscular layer, the panniculus carnosus, which helps wound healing 14. These characteristics render it more difficult to efficiently disrupt the collagen net of the dermis in order to get immune cells out.

In this paper we provide a suitable method for digesting the extracellular matrix that relies on a cocktail of high purity collagenase 1 and 2 and thermolysin. Moreover, since the analysis of skin from different regions requires different experimental procedures, we will show different methods to efficiently obtain skin samples from the footpad, tail, ear and trunk. We will then label the samples in order to evaluate the presence of DCs (MHCII+CD11c+CD45+) Macrophages (CD11b+F4/80+CD45+), CD8+, and CD4+ T cells.

Protocol

연구 승인 : 실험 프로토콜은 Decreto에​​ 따라 건강의 이탈리아 교육부 (로마, 이탈리아)의 승인을 하였다는 N, 27 gennaio 1992 legislativo. (116) "Attuazione 델라 Direttiva N. 609분의 86 / CEE의 본초 디 protezione 글리 animali utilizzati 피니 sperimentali 오 광고 altri 피니 scientifici." 1. 피부 샘플을 얻기 자궁 경부 전위로 마우스를 안락사. 귀: 마우스의 귀 털이없는 부…

Representative Results

신체의 다른 부위에서 피부 표시된 항체와 설명 및 스테인드 프로토콜에 따라 소화했다. 게이팅 전략은 먼저 단봉 (FSC-W 대 FSC-A)와 림프구의 형태 (SSC-A 대 FSC-A)와 세포에서 방송 한 다음 세포 (DAPI- 세포), 게이트를 선택 그림 1에 도시되어있다. 표시 할 때, 조혈 원점에서 CD45 + 세포가 선택됩니다. DC가이 항…

Discussion

우리는 다른 마우스 피부 영역에서 단일 세포 현탁액을 제조하는 방법을 설명 하였다. 우리가 채택 소화 방법은 높은 수율을 제공하지만, 또한 다음 FACS 분석을 위해 근본적인 표면 마커의 발현을 유지 아닙니다.

콜라게나 제 I 및 II와 써모의 칵테일의 사용은,이 방법은 재현성이 높은 만드는 효소 활성의 배치의 차이에 최소 배치를 보장합니다. 다른 공개 방법은 다른 효소 ?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG14593, MFAG13235), the Fondazione Cariplo (Grant 2010-0678 and NANOVAC) and the Fondazione Regionale per la Ricerca Biomedica.

Materials

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 [2.5mg/ml] in dH20 store aliquots at -20°C 
Dnase I Sigma  D4263-1VL resuspend in RPMI 1640 w/o serum [1000u/ml]
Name Company Catalog Number Comments
Materials
Surgical Scissors
Surgical Forceps
35mm petri dishes 
66mm petri dishes
100mm petri dishes
75um Cell Strainers BD 352350
Name Company Clone Label
Antibodies
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
Name Company Catalog Number Comments
Digestion cocktail Dilute in RPMI 5%FBS
LIberase TM  300 ug/ml
DNAse1  50 U/ml

References

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Broggi, A., Cigni, C., Zanoni, I., Granucci, F. Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions. J. Vis. Exp. (110), e52589, doi:10.3791/52589 (2016).

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