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.

研究批准:实验方案得到根据Decreto卫生意大利外交部（意大利，罗马）批准legislativo 27 gennaio 1992年，N。 116"Attuazione德拉Direttiva六百○九分之八十六/ CEE在时珍迪protezione阿布鲁animali utilizzati一个FINI sperimentaliØ广告altri FINI scientificiñ。" 1.获取皮肤样本<ol><li>安乐死颈椎脱位鼠标。 </li><li>耳: <ol><li>切出鼠标的耳朵无毛部分。 </li><li>独立的背部和腹部的侧拉他们除了用钳子。轻轻刮钳耳的内部部分除去任何残留的软骨。 </li></ol></li><li>躯干部皮肤: <ol><li>都与电动剃须刀和精致的化妆水脱毛剃须动物的整皮。 </li><li>切断所需的皮肤样品（约2cm 2至整个动物皮肤）。 <ol><li>如果切断了整个背部和VEN二尖瓣小鼠皮肤，使在小鼠沿着围绕鼠标主体的剃区域的边界背面，然后切的中间垂直切割。 </li><li>轻轻地在肌肤由腹膜和背部肌肉分开，用钳子保持皮肤样本，同时还与分离手术剪刀或手术刀的封闭圆边提示皮肤样本。 </li></ol></li><li>制备含有10ml冰冷的PBS的100毫米细胞培养板。 </li><li>通过浸没在步骤1.3.3准备好的平板在PBS的皮肤样本，并刮用钳子皮肤样本消除皮下脂肪。 </li></ol></li><li>尾巴: <ol><li>切断小鼠的尾部。 </li><li>就用外科剪刀或外科手术刀，从尾基部开始并延伸到尾部的末端的尾部的垂直切割。 </li><li>使用镊子取下从尾部皮肤。抢切的尾巴根部的边缘，向前端按住轻轻一拉用钳子尾体。 </li><li>轻轻刮皮肤样本中删除任何多余的脂肪。 </li></ol></li><li>暴客<ol><li>切出手术剪整个鼠标脚。避免切割任何毛茸茸的皮肤。 </li><li>从脚踝到的数字开始切脚的皮肤纵向。 </li><li>用镊子或用手抓住的同时用钳子皮肤握住脚踝骨头和拉扯皮肤朝向数字仿佛脱下手套。 </li></ol></li></ol> 2.消化<ol><li>准备从原液（如材料清单制备）用下列配方的消化鸡尾酒:酶混合物（参见规格材料清单）300微克/毫升，DNAse1 50单位/毫升。稀释RPMI 5％FBS。 </li><li>砍皮肤样本，用剪刀小块，放入35毫米的培养皿用2毫升消化鸡尾酒（对于躯干部皮肤:皮肤2毫升消化鸡尾酒每2 平方厘米，最高至3ml总数）。 </li><li>孵育在37℃下90分钟。轻轻摇动培养皿孵化期间的1-2倍。 </li><li>倾消化皮肤样品上的70微米的细胞滤网66含有1ml RPMI 1640 5％FBS的毫米的培养皿。 </li><li>用5毫升RPMI 1640 5％FBS的清洗滤网。 </li><li>通过用注射器柱塞细胞过滤挤压样品。 </li><li>冲洗柱塞，培养皿，用20ml的RPMI1640 5％FBS的滤网，转移到50ml管中。 </li></ol> 3.抗体和标记<ol><li>用5毫升PBS中的1％FBS（或BSA）洗所得悬浮液两次。 </li><li>标记与所需的抗体所获得的细胞悬浮液。 <ol><li>重悬的样品在100μl/含有2微克/毫升aCD16 / CD32（克隆2.4G2）的在PBS中的1％FBS的稀释的混合物的样品。 </li><li>为了阻止通过Fc受体的所需抗体的非特异性结合孵育15分钟，4℃。 </li><li>加在表中指出的抗体1。 <ol><li>制备混合稀释PBS中的1％FBS的100微升/样品中的所需抗体在4微克/ ml的浓度。 </li><li>添加在步骤3.2.3.1制备各样品，使得在管中，将有200微升抗体混合物的体积，以2微克/毫升的最终浓度混合100微升。 </li></ol></li><li>孵育在4℃下30分钟，并从光盖。 </li></ol></li><li>用1ml PBS中的1％FBS的，离心洗5分钟，在400 xg离心，弃去300微升PBS中的1％FBS的上清，重悬。 </li><li>添加DAPI以2微克/毫升的最终浓度和孵育在4℃，避光进行10分钟。 </li><li>分析通过FACS获得的样品。 <ol><li>为了分离白细胞，使用下面的门控策略: <ol><li>首先通过门控的DAPI-人口消除任何死细胞。 </li><li>在FSC-A与FSCW¯¯情节选择单线， 如图1所示</strong&gt;。 </li><li>栅极基于尺寸和粒度典型感兴趣的白细胞的细胞。 </li><li>在图1所示的FSC-A地块CD45选择CD45 +细胞。 </li></ol></li><li>通过表面标志物的表达区分白细胞的不同人群如下: <ol><li>确定的DC作为的CD11c + MHC II +细胞。这些可以稍后分析CD207的表达，从CD207-真皮树突区分朗格汉斯细胞和CD207 +真皮树突。 </li><li>检测巨噬细胞作为CD64 + F4 / 80 +细胞。 </li><li>鉴定B细胞CD19 + MHCII +细胞。 </li><li>确定T细胞的CD8 +或CD4 +细胞。 </li></ol></li></ol></li></ol>

<ol><li>Streilein, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circuits+and+signals+of+the+skin-associated+lymphoid+tissues+%28SALT%29%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Circuits and signals of the skin-associated lymphoid tissues (SALT).</a> Journal of Investigative Dermatology. 85 (1 Suppl), 10s-13s (1985).</li>
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<li>Azukizawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Steady+state+migratory+RelB%2B+langerin%2B+dermal+dendritic+cells+mediate+peripheral+induction+of+antigen-specific+CD4%2B+CD25%2B+Foxp3%2B+regulatory+T+cells%5BTitle%5D)+AND+%22European+journal+of+immunology%22%5BJournal%5D)">Steady state migratory RelB+ langerin+ dermal dendritic cells mediate peripheral induction of antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells.</a> European journal of immunology. 41 (5), 1420-1434 (2011).</li>
<li>Idoyaga, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specialized+role+of+migratory+dendritic+cells+in+peripheral+tolerance+induction%5BTitle%5D)+AND+%22The+Journal+of+clinical+investigation%22%5BJournal%5D)">Specialized role of migratory dendritic cells in peripheral tolerance induction.</a> The Journal of clinical investigation. 123 (2), 844-854 (2013).</li>
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<li>Di Meglio, P., Perera, G. K., Nestle, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+multitasking+organ%3A+recent+insights+into+skin+immune+function%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">The multitasking organ: recent insights into skin immune function.</a> Immunity. 35 (6), 857-869 (2011).</li>
<li>Rosenblum, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Response+to+self+antigen+imprints+regulatory+memory+in+tissues%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Response to self antigen imprints regulatory memory in tissues.</a> Nature. 480 (7378), 538-542 (2011).</li>
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The authors declare that they have no competing financial interests.

我们已经描述了用于从不同小鼠皮肤区域制备单细胞悬液的方法。我们通过消化的方法中，不仅使高收率而且还保留了表面标记物的表达，这对于随后的FACS分析的基础。 使用胶原酶I和II和嗜热菌的鸡尾酒，保证最小批量中酶的活性使这种方法高度可再现批次差异。其他公开的方法依赖于不同的酶鸡尾酒但在我们手中，他们给较低的产率，更重要的，是不太可重复的。我们的?...

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.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Supplemented RPMI 1640 medium&amp;nbsp;</td><td></td><td></td><td>RPMI1640 supplemented w/ L-glu, pen-strep w/o beta mercapto ethanol</td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>FBS</td><td></td><td></td><td></td></tr><tr><td>Liberase TM (Enzyme Mix)</td><td>Roche</td><td>5401119001</td><td>resuspend [2.5&amp;nbsp;mg/ml] in dH&lt;sub&gt;2&lt;/sub&gt;O; store aliquots at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>Dnase I</td><td>Sigma&amp;nbsp;</td><td>D4263-1VL</td><td>resuspend in RPMI 1640 w/o serum [1,000&amp;nbsp;U/ml]</td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>Surgical Scissors</td><td></td><td></td><td></td></tr><tr><td>Surgical Forceps</td><td></td><td></td><td></td></tr><tr><td>Surgical Scalpel</td><td></td><td></td><td></td></tr><tr><td>35 mm Petri dishes</td><td></td><td></td><td></td></tr><tr><td>66 mm Petri dishes</td><td></td><td></td><td></td></tr><tr><td>100 mm Petri dishes</td><td></td><td></td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainers</td><td>BD</td><td>352350</td><td></td></tr><tr><td>&lt;strong&gt;Digestion cocktail&lt;/strong&gt;</td><td></td><td></td><td>Dilute in RPMI 5% FBS</td></tr><tr><td>LIberase TM</td><td></td><td></td><td>300 &amp;micro;g/ml</td></tr><tr><td>DNAse1</td><td></td><td></td><td>50 U/ml</td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Clone&lt;/strong&gt;</td><td>&lt;strong&gt;Label&lt;/strong&gt;</td></tr><tr><td>aCD45.2</td><td></td><td>104</td><td>PE-Cy5.5</td></tr><tr><td>aCD11c</td><td></td><td>N418</td><td>PE-Cy7</td></tr><tr><td>CD11b</td><td></td><td>M1/70</td><td>FITC</td></tr><tr><td>F4/80</td><td></td><td>A 3-1</td><td>APC-Cy7</td></tr><tr><td>aCD4</td><td></td><td>Gk1.5</td><td>APC-Cy7</td></tr><tr><td>aCD8</td><td></td><td>53-6.7</td><td>PE</td></tr><tr><td>aCD64</td><td></td><td>X54-5/7.1</td><td>PE-Cy7</td></tr><tr><td>aCD207</td><td></td><td>4C7</td><td>APC</td></tr></tbody></table>

preparation of single-cell suspensions for cytofluorimetric analysis from different mouse skin regions

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.

从身体的不同区域的皮肤根据描述并用所示抗体染色的协议消化。门控策略如图1所示。首先选择汗衫（FSC-A与FSC-W），并与淋巴细胞形态（FSC-A与SSC-A）细胞活细胞（DAPI-细胞），然后门。当指出，从造血系统起源的CD45 +细胞都被选中。 的DC标记与抗CD11c，-MHCII和-CD45抗体，巨噬细胞被标识为F4 / 80 + CD...

Watch this Scientific Journal Video about Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions at JoVE.com

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

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.

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

preparation-single-cell-suspensions-for-cytofluorimetric-analysis

Research

JoVE Journal

Immunology and Infection

18.3K 次观看.  University of Milano-Bicocca. 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. 

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

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

科研

发育生物学

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

免疫与感染

从小鼠肢体肌肉中分离卫星细胞和流式细胞术分析

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

作者聚焦:小鼠肌肉干细胞中的 Cistrome 分析 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

一种用于FACS分离的小鼠卫星细胞的CUT&amp;RUN分析的有效方案

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

生物学

Preparing Mouse Thymic Epithelial Cells for Analysis of Intracellular Molecules

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the thymic cortex induce early T cell development and positive selection of cortical thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla attract positively selected thymocytes from the cortex and establish self-tolerance in T cells. A variety of molecules, including DLL4 and beta5t expressed in cTECs, as well as Aire and CCL21 expressed in mTECs, contribute to thymus function supporting T cell development and selection. Flow cytometric analysis of functionally relevant molecules in cTECs and mTECs is useful to improve our understanding of the biology of TECs, even though current methods for the preparation of single-cell suspensions of TECs can retrieve only a small fraction of TECs (approximately 1% for cTECs and approximately 10% for mTECs) from young adult mouse thymus. Because many of these functionally relevant molecules in TECs are localized within the cells, we describe our protocols for the preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis.

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric AnalysisMechanisms

We describe protocols for the preparation of single-cell suspension of mouse thymic epithelial cells and the staining of intracellular molecules for flow cytometric analysis.

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

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小鼠皮肤成纤维隔离用流式细胞仪

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

从表型分析和人体皮肤淋巴细胞分离

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析