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.

연구 승인 : 실험 프로토콜은 Decreto에​​ 따라 건강의 이탈리아 교육부 (로마, 이탈리아)의 승인을 하였다는 N, 27 gennaio 1992 legislativo. (116) &quot;Attuazione 델라 Direttiva N. 609분의 86 / CEE의 본초 디 protezione 글리 animali utilizzati 피니 sperimentali 오 광고 altri 피니 scientifici.&quot; 1. 피부 샘플을 얻기 <ol><li> 자궁 경부 전위로 마우스를 안락사. </li><li> 귀: <ol><li> 마우스의 귀 털이없는 부분을 잘라. </li><li> 집게로 떨어져 당겨 분리 지느러미와 복부 측면. 부드럽게 집게로 귀 안쪽 부분을 긁어로 남아있는 연골을 제거합니다. </li></ol></li><li> 트렁크 피부 : <ol><li> 전기 면도기와 섬세한 제모 로션 두 동물의 전체 피부를 면도. </li><li> (2cm 2의 전체 동물의 피부까지) 원하는 피부 샘플을 잘라. <ol><li> 전체 지느러미를 절단하는 경우와 VENTRAL 마우스 피부는 다시 다음 마우스 본체 주변의 면도 영역의 국경을 따라 절단 한 마우스의 중앙에 수직 절단을합니다. </li><li> 조심스럽게 수술 가위 나 메스의 폐쇄 라운드 예리하게 팁을 피부 샘플을 분리하면서 집게로 여전히 피부 샘플을 유지, 복막 다시 근육으로부터 피부를 분리합니다. </li></ol></li><li> 빙냉 PBS 10 ㎖를 함유하는 100mm 세포 배양 플레이트를 준비한다. </li><li> 단계 1.3.3에서 제조 한 접시에 PBS의 피부 샘플을 침수와 집게로 피부 샘플을 긁어하여 피하 지방을 제거합니다. </li></ol></li><li> 꼬리: <ol><li> 마우스의 꼬리를 잘라. </li><li> 수술 용 가위 외과 메스, 꼬리베이스에서 시작하여 꼬리의 끝 부분에 도달와 꼬리에 수직 절단합니다. </li><li> 꼬리으로부터 피부를 분리 집게를 사용합니다. 꼬리의 기지에서 절단의 가장자리를 잡고하고 채 끝을 향해 조심스럽게 잡아 당겨집게와 꼬리의 몸. </li><li> 부드럽게 피부 샘플을 긁어에 의해 어떤 여분의 지방을 제거합니다. </li></ol></li><li> 노상 강도 <ol><li> 수술 가위로 전체 마우스 발을 잘라. 모든 털이 피부를 절단 방지. </li><li> 숫자의 시작 발목에서 길이 방향 다리의 피부를 잘라. </li><li> 집게로 피부를 잡는 동안 집게 또는 손으로 발목 뼈를 잡고 장갑을 벗고 것처럼 자리를 향해 피부를 잡아 당깁니다. </li></ol></li></ol> 2. 소화 <ol><li> 효소 믹스 (사양 재료 목록 참조) 300 μg의 / ㎖, DNAse1 50 U / ㎖ 다음 레시피 (재료 목록에서와 같이 제조) 원액에서 소화 칵테일을 준비합니다. RPMI 5 % FBS에 희석. </li><li> (트렁크 피부 : 피부의이 소화 칵테일 ml의 모든 2cm 2, 최대 3 ㎖ 총) 소화 칵테일 2 ㎖와 35mm 페트리 접시에 작은 조각으로 가위로 피부 샘플을 잘라. </li><li>90 분 동안 37 ° C에서 품어. 조심스럽게 배양시 배양 접시를 1-2 번 흔들어. </li><li> RPMI1640 5 % FBS 1 ㎖를 함유하는 66mm 페트리 접시에서 70 μm의 셀 스트레이너에 소화 된 피부 샘플을 붓는다. </li><li> RPMI 1640 5 % FBS의 5 ㎖로 스트레이너 씻으십시오. </li><li> 주사기 플런저 셀 스트레이너를 통해 샘플을 짠다. </li><li> 세척 플런저, 페트리 접시와 RPMI1640 5 % FBS 20 ㎖와 스트레이너, 50 ㎖ 튜브에 전송할 수 있습니다. </li></ol> 3. 항체 및 라벨링 <ol><li> PBS 1 % FBS (또는 BSA)을 5 ml로 두 번 얻어진 현탁액을 씻으십시오. </li><li> 원하는 항체 얻어진 세포 현탁액 레이블. <ol><li> 100 μL에 재현 탁 샘플 / PBS 1 % FBS에 희석 aCD16 / CD32 (클론 2.4G2) 2 ㎍ / ml를 함유하는 혼합물의 샘플. </li><li> Fc 수용체를 통해 원하는 항체의 비특이적 결합을 차단하기 위해 4 ℃에서 15 분 동안 인큐베이션. </li><li> 표에 표시된 항체를 추가1. <ol><li> 4 μg / ml의 농도 PBS, 1 % FBS 100 ㎕ / 샘플의 원하는 항체를 희석 혼합하여 준비한다. </li><li> 튜브에서 2 μg / ML의 최종 농도로 항체 혼합물의 200 μL의 부피가되도록 각 시료에 3.2.3.1 단계에서 제조 된 혼합물 100 ㎕를 추가한다. </li></ol></li><li> 4 ° C에서 30 분 동안 품어 빛에서 다룹니다. </li></ol></li><li> 400 XG에서 5 분 동안 PBS 1 % FBS 1 ㎖, 원심 분리로 세척 PBS 1 % FBS 300 μL의 상층 액과에 resuspend 폐기합니다. </li><li> 2 μg / ML의 최종 농도에서 DAPI를 추가하고 10 분 동안 빛으로부터 보호, 4 ° C에서 배양한다. </li><li> FACS에 의해 얻어진 샘플을 분석 할 수 있습니다. <ol><li> 백혈구를 분리하기 위해, 다음 게이팅 전략을 사용 <ol><li> 먼저 DAPI- 인구에 게이팅에 의해 죽은 세포를 제거한다. </li><li> 그림 1에 나타낸 바와 같이, FSC의 W 플롯 대 FSC-A에서 선택 단봉 </strong&gt;. </li><li> 게이트 주목 백혈구의 전형적인 크기와 입도에 기초하여 상기 셀. </li><li> 그림 1에 나타낸 바와 같이 FSC-A CD45 플롯에서 CD45 + 세포를 선택합니다. </li></ol></li><li> 다음과 같이 표면 마커의 발현에 의해 백혈구의 다양한 인구를 구별 : <ol><li> 중 CD11c + MHC II + 세포로 수지상 세포를 식별합니다. 이들은 나중에 CD207- 피부 수지상에서 랑게르한스 세포 및 피부 CD207 + 수지상 세포를 구별하는 CD207의 발현에 대해 분석 할 수있다. </li><li> CD64 + F4 / 80 + 세포로서 대 식세포를 식별합니다. </li><li> CD19 + MHCII + 세포와 같은 B 세포를 식별합니다. </li><li> CD8 + 또는 CD4 + 세포와 같은 T 세포를 식별합니다. </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

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 ?...

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.

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...

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.

다른 마우스 피부 지역에서 Cytofluorimetric 분석을위한 단일 세포 현탁액의 준비

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 조회수.  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. 

비디오: 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.

FACS에 의해 쥐 피부 섬유 아세포 격리

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

연구

발생학

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.

등쪽 마우스 피부에서 헤어 난포 줄기 세포와 표피 각질 형성 세포를 분리

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.

질량 세포 측정에 의한 세포 단백질 발현의 세포 주기 특이적 분석을 위한 마우스 피부 각질 세포의 분리 및 염색

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.

표현형 분석 및 대한 인간의 피부에서 림프구 분리<em&gt; 전의 VIVO</em&gt; 세포 배양

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

면역학 및 감염병학

Satellite Cell from Mouse Limb Muscles and Flow Cytometry Analysis (마우스 사지 근육에서 위성 세포 분리 및 유세포 분석)

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.

저자 스포트라이트: 마우스 근육 줄기 세포의 Cistrome 분석 

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 분리 마우스 위성 세포의 CUT&amp;RUN 분석을 위한 효율적인 프로토콜

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.

형광 활성 세포 정렬 (FACS)에 의해 신선한 조직으로부터 정상 및 암 관련 섬유 아세포의 분리

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

생물학

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 프로토콜 전체 마운트 피부 준비 단일 셀 해상도에서 단백질 발현을 시각화합니다

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

다른 마우스 피부 지역에서 Cytofluorimetric 분석을위한 단일 세포 현탁액의 준비

요약

더 많은 비디오 탐색

이 비디오의 챕터

FACS에 의해 쥐 피부 섬유 아세포 격리

등쪽 마우스 피부에서 헤어 난포 줄기 세포와 표피 각질 형성 세포를 분리

질량 세포 측정에 의한 세포 단백질 발현의 세포 주기 특이적 분석을 위한 마우스 피부 각질 세포의 분리 및 염색

표현형 분석 및 대한 인간의 피부에서 림프구 분리

FACS 분리 마우스 위성 세포의 CUT&RUN 분석을 위한 효율적인 프로토콜

FACS 분리 마우스 위성 세포의 CUT&RUN 분석을 위한 효율적인 프로토콜

FACS 분리 마우스 위성 세포의 CUT&RUN 분석을 위한 효율적인 프로토콜

형광 활성 세포 정렬 (FACS)에 의해 신선한 조직으로부터 정상 및 암 관련 섬유 아세포의 분리

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

CUBIC 프로토콜 전체 마운트 피부 준비 단일 셀 해상도에서 단백질 발현을 시각화합니다