Name: preparation of single-cell suspensions for cytofluorimetric analysis from different mouse skin regions
Uploaded: 2016-04-20T13:00:00.000+00:00
Duration: 5 min 58 s
Description: Watch this Scientific Journal Video about Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions at JoVE.com

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.

研究の承認：実験プロトコルはDecretoに応じた保健のイタリア省（ローマ、イタリア）によって承認された27 gennaio 1992 legislativo、n個。 116「AttuazioneデッラDirettivaのn。609分の86 / CEEでマテリアディprotezioneデッリanimali utilizzati Aフィニsperimentali O広告altriフィニscientifici。」 1.皮膚サンプルを入手<ol><li>頸椎脱臼によりマウスを安楽死させます。 </li><li>耳： <ol><li>マウスの耳の毛のない部分を切り取ります。 </li><li>鉗子でそれらを引き離すことにより、独立した背と腹側。静かにピンセットで耳の内側の部分をこすることによって、残りの軟骨を取り除きます。 </li></ol></li><li>トランク皮膚： <ol><li>電気かみそりで、繊細な脱毛ローションで両方の動物の皮膚全体を剃ります。 </li><li> （2 cm 2で全動物の皮膚まで）所望の皮膚サンプルを遮断。 <ol><li>全体の背側を切断した場合とVENクトルのマウスの皮膚は、バックした後、マウス本体の周りの毛を剃った領域の境界線に沿って切断し、マウスの真ん中の縦のカットを行います。 </li><li>静かに手術用ハサミやメスの閉じラウンドエッジの先端で皮膚のサンプルを分離しながら、鉗子でまだ皮膚サンプルを保ち、腹膜や背中の筋肉から皮膚を分離します。 </li></ol></li><li>氷冷PBSの10ミリリットルを含む100ミリメートル細胞培養プレートを準備します。 </li><li>ステップ1.3.3で製造したプレートにPBSで皮膚サンプルを浸漬し、鉗子で皮膚試料をこすることによって皮下脂肪を除去します。 </li></ol></li><li>尾： <ol><li>マウスの尾を切り落としました。 </li><li>尾の基部から始まり、尾の先端に到達し、手術用はさみまたは外科メスで尾の垂直カットを行います。 </li><li>尾から皮膚を分離するために鉗子を使用してください。尾の基部にカットのエッジをつかみ、そっと押さえながら先端に向かって引っ張ります鉗子で尾の体。 </li><li>優しく皮膚試料をこすることによって、任意の余分な脂肪を取り除きます。 </li></ol></li><li>追いはぎ<ol><li>手術用ハサミでマウス全体の足を切り取ります。任意の有毛皮膚を切断回避。 </li><li>数字の先頭に縦方向に足首から足の皮膚をカット。 </li><li>鉗子で皮膚をつかんながら鉗子でまたは手で足首の骨を持ち、手袋を脱いでいるかのように数字に向かって皮膚を引っ張ります。 </li></ol></li></ol> 2.消化<ol><li>酵素ミックス（仕様のための材料リストを参照してください）​​300μgの/ mlの、DNAse1 50 U / mlの次のレシピ（材料リストのように調製）ストック溶液から消化カクテルを準備します。 RPMI 5％FBS中で希釈します。 </li><li> （：消化カクテルの2ミリリットル皮膚のあらゆる2 cm 2で、最大3 mlの全トランク肌用）消化カクテルの2ミリリットルと35ミリメートルペトリ皿に小片にハサミで皮膚サンプルをみじん切りに。 </li><li>90分間37℃でインキュベートします。ゆっくりインキュベーション中のペトリ皿に1-2回振ります。 </li><li> RPMI1640 5％FBSの1ミリリットルを含む66ミリメートルペトリ皿に70μmのセルストレーナーに消化された皮膚試料を注ぎます。 </li><li> RPMI 1640の5ミリリットルの5％FBSでストレーナーを洗ってください。 </li><li>シリンジプランジャと細胞ストレーナーを通して試料を絞ります。 </li><li>フラッシュプランジャー、RPMI1640 5％FBSの20ミリリットルとペトリ皿とストレーナ、50ミリリットルチューブに移します。 </li></ol> 3.抗体とラベリング<ol><li> PBS、1％FBS（またはBSA）の5ミリリットルで二回得られた懸濁液を洗​​ってください。 </li><li>所望の抗体で得られた細胞懸濁液にラベルを付けます。 <ol><li> 100μlのサンプルを再懸濁/ PBS、1％FBS中で希釈したaCD16 / CD32（クローン2.4G2）を2μg/ mlを含むミックスのサンプル。 </li><li> Fc受容体を介して、所望の抗体の非特異的結合をブロックするために4℃で15分間インキュベートします。 </li><li> 表に示した抗体を追加します。1。 <ol><li> 4μg/ mlの濃度でPBS、1％FBSを100μl/サンプル中の所望の抗体を希釈ミックスを調製します。 </li><li>チューブに、2μgの/ mlの最終濃度で抗体混合物200μlの容量が存在することになるように、各サンプルにステップ3.2.3.1で準備ミックス100μlのを追加します。 </li></ol></li><li> 4℃で30分間インキュベートし、光から覆います。 </li></ol></li><li> PBS、1％FBSの1ミリリットル、400×gで5分間の遠心分離で洗浄し、PBS、1％FBSの300μlの上清および再懸濁を捨てます。 </li><li> 2μg/ mlの最終濃度でDAPIを加え、10分間、光から保護し、4℃でインキュベートします。 </li><li> FACSによって得られた試​​料を分析します。 <ol><li>白血球を単離するために、以下のゲーティング戦略を使用します。 <ol><li>まずDAPI-集団に対してゲートすることによって、任意の死細胞を排除します。 </li><li> 図1に示すように、FSCのWプロット対FSC-Aにシングレットを選択</strong&gt;。 </li><li>ゲート関心の白血球の典型的な大きさと細かさに基づいてセル。 </li><li> 図1に示すようにFSC-CD45プロットでCD45 +細胞を選択します。 </li></ol></li><li>次のように、表面マーカーの発現によって、白血球の異なる集団を区別する。 <ol><li> CD11c + MHC II +細胞としてDCを識別します。これらは、後でCD207-皮膚樹状細胞からのランゲルハンス細胞及びCD207 +皮膚樹状細胞を区別するためにCD207の発現について分析することができます。 </li><li> CD64 + F4 / 80 +細胞としてマクロファージを識別します。 </li><li> CD19 + MHCII +細胞などのB細胞を識別します。 </li><li> CD8 +またはCD4 +細胞のようなT細胞を識別します。 </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

我々は、異なるマウス皮膚領域からの単一細胞懸濁液を調製するための方法を記載しています。我々が採用した消化の方法は、高い収率を与えるだけでなく、その後のFACS分析のために基本的な表面マーカーの発現を維持するだけでなく。 コラゲナーゼIおよびIIおよびサーモリシンのカクテルの使用は、この方法は、再現性の高い作り酵素活性のバッチの違いに最小バッ?...

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.

体のさまざまな部位からの皮膚は、標識した抗体を用い説明し、染色されたプロトコルに従って消化しました。ゲーティング戦略は、まずシングレット（FSC-W対FSC-A）にし、リンパ球の形態（SSC-A対FSC-A）を持つ細胞でライブ後、細胞（DAPI-細胞）、ゲートを選択し、図1に示されています。示された場合に、造血起源のCD45 +細胞が選択されます。 </p...

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.

別のマウス皮膚の領域から細胞蛍光分析のための単一細胞懸濁液の調製

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

18.2K ビュー.  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. 

ビデオ: 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.

FACSによるマウス皮膚線維芽細胞の分離

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

発生生物学

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.

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

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 ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

質量細胞血症による細胞タンパク質発現の細胞周期特異的解析におけるマウス皮膚ケラチノサイトの単離と染色

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

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.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

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・感染学

マウス四肢筋からのサテライト細胞単離とフローサイトメトリー解析

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.

著者スポットライト:マウス筋幹細胞におけるCistrome解析 

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).

FACS単離マウスサテライト細胞のCUT&amp;RUN解析のための効率的なプロトコール

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.

蛍光活性化細胞選別（FACS）によって新鮮な組織からの正常および癌関連線維芽細胞の分離

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プロトコルは、全マウントスキン準備中の単一細胞の解像度でのタンパク質発現を可視化

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

別のマウス皮膚の領域から細胞蛍光分析のための単一細胞懸濁液の調製

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FACSによるマウス皮膚線維芽細胞の分離

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

質量細胞血症による細胞タンパク質発現の細胞周期特異的解析におけるマウス皮膚ケラチノサイトの単離と染色

表現型分析のためのヒト皮膚からリンパ球単離と

FACS単離マウスサテライト細胞のCUT&RUN解析のための効率的なプロトコール

FACS単離マウスサテライト細胞のCUT&RUN解析のための効率的なプロトコール

FACS単離マウスサテライト細胞のCUT&RUN解析のための効率的なプロトコール

蛍光活性化細胞選別（FACS）によって新鮮な組織からの正常および癌関連線維芽細胞の分離

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

CUBICプロトコルは、全マウントスキン準備中の単一細胞の解像度でのタンパク質発現を可視化