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.

Studienzulassung: Die experimentellen Protokolle wurden vom italienischen Ministerium für Gesundheit genehmigt (Rom, Italien) nach dem Decreto legislativo 27 novembre 1992, n. 116 &quot;attuazione della Direttiva n. 86/609 / EWG in materia di Protezione degli animali utilizzati a fini sperimentali o ad altri fini scientifici.&quot; 1. Gewinnung Hautproben <ol><li> Euthanize die Maus durch Genickbruch. </li><li> Ohr: <ol><li> Schneiden Sie den unbehaarten Teil der Ohren der Maus aus. </li><li> Separate dorsalen und ventralen Seite durch sie auseinander mit einer Pinzette herausziehen. Entfernen Sie alle verbleibenden Knorpeln durch sanft den inneren Teil der Ohren mit einer Pinzette Schaben. </li></ol></li><li> Trunk Haut: <ol><li> Rasieren Sie die ganze Haut des Tieres sowohl mit einem elektrischen Rasierer und mit zarten Enthaarungslotion. </li><li> Schneiden Sie die gewünschte Hautprobe (von 2 cm 2 bis zur gesamten Tierhaut) aus. <ol><li> Wenn die gesamte Rücken abschneidet und ventrale Mäusehaut, einen vertikalen Schnitt in der Mitte der Maus entlang der Grenzen der rasierten Bereichen rund um die Maus Körper zurück geschnitten und dann machen. </li><li> Vorsichtig trennen die Haut aus dem Peritoneum und Rückenmuskulatur, immer noch mit der Pinzette die Hautprobe zu halten, während die Hautprobe mit geschlossenen runden kantigen Spitzen einer chirurgischen Schere oder einem Skalpell trennt. </li></ol></li><li> Bereiten Sie eine 100 mm Zellkulturplatte mit 10 ml eiskaltem PBS enthält. </li><li> Beseitigen von subkutanem Fett durch Eintauchen der Hautprobe in PBS in der Platte, hergestellt in Schritt 1.3.3 und Abkratzen der Hautprobe mit einer Pinzette. </li></ol></li><li> Schwanz: <ol><li> Schneiden Sie den Schwanz der Maus ab. </li><li> Machen Sie einen vertikalen Schnitt auf den Schwanz mit einer chirurgischen Schere oder chirurgische Skalpell aus der Schwanzbasis beginnt und an der Spitze des Schwanzes zu erreichen. </li><li> Verwenden einer Pinzette, um die Haut vom Schwanz zu lösen. Besorgen Sie sich die Kante des Schnitts an der Basis des Schwanzes und ziehen Sie vorsichtig die Spitze zu, während die HalteKörper des Schwanzes mit einer Pinzette. </li><li> Entfernen Sie überschüssiges Fett durch die Hautprobe sanft kratzen. </li></ol></li><li> Wegelagerer <ol><li> Schneiden Sie die gesamte Maus Fuß mit chirurgischen Schere. Vermeiden jede behaarter Haut zu schneiden. </li><li> Schneiden Sie die Haut des Fußes in Längsrichtung vom Knöchel bis zum Beginn der Ziffern. </li><li> Halten Sie die Knöchel mit einer Pinzette oder mit der Hand, während die Haut mit einer Pinzette greifen und ziehen Sie die Haut in Richtung auf die Ziffern, als ob, die ein Handschuh aus. </li></ol></li></ol> 2. Verdauung <ol><li> Bereiten Sie die Verdauung Cocktail aus der Stammlösung (hergestellt wie in der Materialliste) mit folgendem Rezept: Enzymmischung (siehe Materialliste für Spezifikationen) 300 ug / ml, DNAse1 50 U / ml. Man verdünnt in RPMI 5% FBS. </li><li> Hacken Hautproben mit einer Schere in kleine Stücke in eine 35 mm Petrischale mit 2 ml Digestion Cocktail (Für Stamm Haut: 2 ml Verdauung Cocktail alle 2 cm 2 der Haut, bis zu 3 ml insgesamt). </li><li>für 90 min bei 37 ° C inkubieren. Schütteln Sie die Petrischale 1-2 mal während der Inkubation. </li><li> Gießen verdaute Hautproben auf einem 70 &amp; mgr; m Zellsieb in 66 mm Petrischale mit 1 ml RPMI1640 5% FBS. </li><li> Waschen Sie das Sieb mit 5 ml RPMI 1640 5% FBS. </li><li> Drücken Sie die Probe durch die Zelle Sieb mit einem Spritzenkolben. </li><li> Flush Kolben, Petrischale und Sieb mit 20 ml RPMI1640 5% FBS, Transfer in 50-ml-Tube. </li></ol> 3. Antikörper und Labeling <ol><li> Waschen der erhaltenen Suspension zweimal mit 5 ml PBS, das 1% FBS (oder BSA). </li><li> Beschriften Sie die erhaltene Zellsuspension mit den gewünschten Antikörper. <ol><li> Resuspendieren der Proben in 100 ul / Probe einer Mischung enthaltend 2 &amp; mgr; g / ml aCD16 / CD32 (Klon 2.4G2), verdünnt in PBS 1% FBS. </li><li> 15 min Inkubieren bei 4 ° C, um nicht-spezifische Bindung der gewünschten Antikörper via Fc-Rezeptoren zu blockieren. </li><li> Fügen Sie die angegebenen Antikörper in der Tabelle1. <ol><li> Bereiten einer Mischung die gewünschten Antikörper in 100 &amp; mgr; l / Probe von PBS 1% FBS in einer Konzentration von 4 ug / ml verdünnt. </li><li> Füge 100 &amp; mgr; l des in Schritt hergestellten Mischung 3.2.3.1 zu jeder Probe, so dass in dem Rohr, wird es in einer Endkonzentration von 2 &amp; mgr; g / ml mit einem Volumen von 200 &amp; mgr; l der Antikörpermischung sein. </li></ol></li><li> Inkubieren für 30 min bei 4 ° C und die Abdeckung von Licht. </li></ol></li><li> Waschen mit 1 ml PBS, das 1% FBS, Zentrifuge für 5 Minuten bei 400 · g, den Überstand verwerfen und resuspendieren in 300 ul PBS, das 1% FBS. </li><li> Hinzufügen DAPI in einer Endkonzentration von 2 &amp; mgr; g / ml und Inkubation bei 4 ° C, geschützt vor Licht für 10 min. </li><li> Analysieren Sie die Proben durch FACS erhalten. <ol><li> Um Leukozyten zu isolieren, verwenden Sie die folgende Gating-Strategie: <ol><li> Zuerst beseitigen alle toten Zellen durch auf der DAPI- Bevölkerung Gating. </li><li> Wählen Sie Singuletts im FSC-A gegen FSC W Plot , wie in Abbildung 1 angegeben </strong&gt;. </li><li> Tor die darauf basierenden Zellen nach Größe und Granularität typisch für die Leukozyten von Interesse. </li><li> Wählen Sie CD45 + Zellen in der FSC-A CD45 Plot wie in Abbildung 1 dargestellt. </li></ol></li><li> Unterscheiden die verschiedenen Populationen von Leukozyten durch die Expression von Oberflächenmarkern, wie folgt: <ol><li> Identifizieren DCs als CD11c + MHC II + Zellen. Diese können später für die Expression von CD207 analysiert werden, um zu unterscheiden, Langerhans&#39;schen Zellen und CD207 + Dermal DCs von CD207- Dermal DCs. </li><li> Identifizieren Makrophagen als CD64 + F4 / 80 + Zellen. </li><li> Identifizieren B-Zellen als CD19 + MHCII + Zellen. </li><li> Identifizieren T-Zellen als CD8 + oder CD4 + Zellen. </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Wir haben ein Verfahren zur Herstellung von Einzelzellsuspensionen aus verschiedenen Maushautregionen beschrieben. Verfahren nach Verdauung wir angenommen haben, verursacht nicht nur hohe Ausbeuten, sondern bewahrt auch die Expression von Oberflächenmarkern, die für die nachfolgende FACS-Analyse von grundlegender Bedeutung ist. Die Verwendung eines Cocktails von Kollagenase I und II und Thermolysin, garantiert minimale Charge zu Charge unterschiedliche Enzymaktivität macht dieses Verfahre...

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.

Haut von anderen Bereich des Körpers wurde gemäß dem Protokoll, verdaut mit den angegebenen Antikörpern beschrieben und fleckig. Die Gating - Strategie ist in Abbildung 1. Wählen Sie zuerst lebende Zellen (DAPI- Zellen), dann Tor auf Singuletts (FSC-A gegen FSC-W) und auf Zellen mit Lymphozytenmorphologie (FSC-A vs. SSC-A) dargestellt. Wenn angegeben, werden CD45 + Zellen aus hämatopoetischen Ursprungs ausgewählt. <p class="jove_content" fo:keep-tog...

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.

Herstellung von Einzelzellsuspensionen für Cytofluorimetric Analyse von verschiedenen Mäusehaut Regionen

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 Aufrufe.  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. 

Serie: 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.

Murine Hautfibroblasten-Isolation durch FACS

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

Forschung

Entwicklungsbiologie

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.

Isolieren von Haarfollikelstammzellen und epidermalen Keratinozyten von Dorsal Mäusehaut

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.

Isolierung und Färbung der Maushaut Keratinozyten für Zellzyklus spezifische Analyse der zellulären Proteinexpression durch Massenzytometrie

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.

Lymphocyte Isolierung von menschlicher Haut für phänotypische Analyse und<em&gt; Ex Vivo</em&gt; Zellkultur

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

Immunologie und Infektion

Isolierung von Satellitenzellen aus Gliedmaßenmuskeln der Maus und Durchflusszytometrie-Analyse

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.

Autor im Rampenlicht: Cistrome-Analyse in Muskelstammzellen der Maus 

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).

Ein effizientes Protokoll für die CUT&amp;RUN-Analyse von FACS-isolierten Maus-Satellitenzellen

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.

Isolation von Normal und Krebs-assoziierte Fibroblasten von Fresh Gewebe durch 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 ...

Biologie

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 Protokoll Visualisiert Expression Protein bei Single Cell Resolution in Ganze Berge Vorbereitung der Haut

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

Herstellung von Einzelzellsuspensionen für Cytofluorimetric Analyse von verschiedenen Mäusehaut Regionen

Zusammenfassung

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Isolieren von Haarfollikelstammzellen und epidermalen Keratinozyten von Dorsal Mäusehaut

Isolierung und Färbung der Maushaut Keratinozyten für Zellzyklus spezifische Analyse der zellulären Proteinexpression durch Massenzytometrie

Lymphocyte Isolierung von menschlicher Haut für phänotypische Analyse und

Ein effizientes Protokoll für die CUT&RUN-Analyse von FACS-isolierten Maus-Satellitenzellen

Ein effizientes Protokoll für die CUT&RUN-Analyse von FACS-isolierten Maus-Satellitenzellen

Ein effizientes Protokoll für die CUT&RUN-Analyse von FACS-isolierten Maus-Satellitenzellen

Isolation von Normal und Krebs-assoziierte Fibroblasten von Fresh Gewebe durch Fluorescence Activated Cell Sorting (FACS)

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

CUBIC Protokoll Visualisiert Expression Protein bei Single Cell Resolution in Ganze Berge Vorbereitung der Haut