다른 마우스 피부 지역에서 Cytofluorimetric 분석을위한 단일 세포 현탁액의 준비

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.