Preparação de uma única célula suspensões para Análise de citometria de fluxo de diferentes regiões da pele do rato
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
Representative Results
Discussion
Descrevemos um método para a preparação de suspensões unicelulares a partir de regiões diferentes da pele do rato. O método de digestão adoptámos, não só dá rendimentos elevados mas também preserva a expressão de marcadores de superfície, que é fundamental para a análise de FACS subsequente.
A utilização de uma mistura de colagenase I e II e termolisina, garante lote mínimo de diferenças de lote na actividade da enzima, tornando este método altamente reprodutível. Outro…
Declarações
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 |
Referências
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