We thank Anastasia Bannwarth for providing excellent technical assistance. We thank the IGBMC animal house facility, the cell culture, the Mouse Clinical Institute (ICS, Illkirch, France), the imaging, the electron microscopy, the flow cytometry, and the GenomEast platform, a member of the &#39;France G&#233;nomique&#39; consortium (ANR-10-INBS-0009).
This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002) and by SFRI-STRAT&#39;US project (ANR 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. Additional funding was delivered by INSERM, CNRS, Unistra, IGBMC, Agence Nationale de la Recherche (ANR-16-CE11-0009, AR2GR), AFM-T&#233;l&#233;thon strategic program 24376 (to D.D.), INSERM young researcher grant (to D.D.), ANR-10-LABX-0030-INRT, and a French State fund managed by the ANR under the frame program Investissements d&#39;Avenir (ANR-10-IDEX-0002-02). J.R. was supported by the Programme CDFA-07-22 from the Universit&#233; franco-allemande and Minist&#232;re de l&#39;Enseignement Sup&#233;rieur de la Recherche et de l&#39;Innovation, and K.G. by the Association pour la Recherche &#224; l&#39;IGBMC (ARI).

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

<ol>
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The authors declare that they have no competing financial interests.

The present study reports a standardized, reliable, and easy-to-perform method for the isolation and culture of mouse satellite cells, as well as the assessment of transcriptional regulation by the CUT&#38;RUN method.
This protocol involves several critical steps. The first is muscle disruption and fiber digestion to ensure a high number of collected cells. Despite the increased enzyme concentration, more living cells were obtained than using Protocol 1. Satellite cells express a specific pattern of various membrane proteins. To increase the stringency of our sorting, we used a combination of previously described negative (CD31, TER119, CD45, and CD11b) and positive (CD34, ITGA7, and CXCR4) satellite cell markers30,31. Using this strategy, an average of 1% of living putative satellite cells was obtained. This outcome is in the range of what was expected, since satellite cells represent 2%-7% of muscle cell population at adulthood in mice32. As a control for the satellite cell selection markers, CD34+/ITGA7-/CXCR4- cells were isolated. Immunofluorescent staining demonstrated that, whereas CD34+/ITGA7-/CXCR4- cells did not express PAX7, 70% of CD34+/ITGA7+/CXCR4+ sorted cells were positive for this satellite cell-specific marker, thereby demonstrating that the FACS isolated population corresponds&#160;to satellite cells. In addition, 70% of satellite cells grown in myogenic medium for 7 days were PAX7-/DMD+, as shown by immunostaining, and formed an elongated multinucleated myofiber, demonstrating that isolated satellite cells conserved their stemness potential.
The major limitation in sample preparation might be the use of a high concentration of enzymes, which results in a large amount of dissociated biological material, but may contribute to increased cell death. To resolve this issue, an assay requiring lower enzyme quantity was tested. In this context, Liberase TL, a purified form of the traditional collagenase33, was tested. Although digestion was apparently more efficient with this enzyme, the amount of cell debris remained elevated, and the cell viability was slightly lowered. These observations are in agreement with previous reports that compared Liberase-mediated tissue digestion to this with recombinant collagenase or custom collagenase24,25. In addition, even though the proportion of endothelial and immune cells was similar between collagenase and Liberase digestion, the percentage of satellite cells was lower in the Liberase-processed sample, overall showing that Liberase is not recommended for satellite cell isolation.&#160;The use of this enzyme is suitable for the phenotypic and quantitative analysis of immune cells and could eventually be recommended in the context of muscle and/or other tissues. This is in concordance with what has been reported on Liberase TL as the most suited to effectively isolate viable immune cells34,35,36.
Another potential issue is the stress caused by both dissociating and sorting satellite cells. However, the absence of H3K27ac and the low H3K4me2 levels at the promoter region of stress response genes identified by single-cell RNA sequencing in mouse satellite cells26 show that the procedure is mild enough not to induce the stress response transcriptional repertoire. Other limitations to be noted are the selected antigens that remain empiric. However, studies show a high overlap between unique surface marker combinations that are enriched for skeletal muscle satellite cells30,31.
An additional critical step is the purification of nuclei from sorted cells. For this, the sorting speed is maintained low in order not to damage the cells, but fast enough that they are not stored for too long in FACS buffer. Indeed, CUT&#38;RUN nuclei are not fixed, and a longer incubation time might influence the read obtained from the protocol. A typical preparation from the two hind limbs of one mouse allows CUT&#38;RUN with one antibody and one IgG control, with an amount of 2.5 ng of chromatin for each.
CUT&#38;RUN experiments, designed to assess transcription factor binding and epigenetic modifications, provided strong and robust read signals for H3K4me2 and H3K27ac. The high read levels of H3K4me2 and H3K27ac on genes that are known to be expressed in satellite cells, compared to genes expressed in immune or endothelial cells as well as in myofibers, showed that the signal was amplified predominantly from the satellite cells nuclei. In addition, this protocol made the identification of the cistrome of the AR in muscle stem cells possible, which to date remains a technical issue inherent to the poor quality of mouse AR antibodies and the low AR expression levels in cells that are not highly androgen-sensitive, such as epithelial prostate cells. The data presented here unveiled AR-bound regions with high confidence peak scores. As only one replicate was originally assessed per antibody, the robustness of our datasets will be improved by adding at least two additional biological replicates per condition. However, AR known target genes and respective histone marks, originally described as highly expressed and/or easily detectable in other tissue contexts such as the prostate, present lower expression levels and are very challenging to detect in satellite cells.
One limitation of the CUT&#38;RUN approach is that it is performed in unfixed cells. Thus, we might have missed some AR targets because of the labile nature of the interactions between transcription factors and their binding site. Similarly, some post-translational modifications, such as acetylation, can also be rather labile. Therefore, including a light fixation step with formaldehyde or paraformaldehyde, after FACS sorting, and before isolating the nuclei, could be considered to stabilize the protein/DNA interactions and histone modifications.
Although this paper shows that CUT&#38;RUN assays can be conducted on FACS-isolated satellite cells, other analyses that use non-fixed chromatin such as ATAC-seq might also be performed. In addition, even though here muscles were harvested in basal physiological conditions, this protocol may be applied to pathological contexts such as injury or aging. All these protocol uses will allow a detailed understanding of gene regulatory mechanisms in satellite cells, and may help in understanding how these mechanisms might be altered by pathophysiological conditions.
In summary, this low-cost and time-efficient protocol provides an effective experimental setting to study transcription factor recruitment and chromatin landscape in skeletal muscle precursor cells. Chromatin prepared by this protocol has provided the first genome-wide analysis of AR cistrome in satellite cells and will facilitate future studies on gene regulation.

Skeletal striated muscles represent on average 40% of the weight of the total human body1. Muscle fibers exhibit a remarkable capacity for regeneration upon injury, which is described by the fusion of newly formed myocytes and the generation of new myofibers that replace the damaged ones2. In 1961, Alexander Mauro reported a population of mononuclear cells that he termed as satellite cells3. These stem cells express the transcription factor paired box 7 (PAX7), and are located between the basal lamina and the sarcolemma of muscle fibers4. They were reported to express the cluster of differentiation 34 (CD34; a hematopoietic, endothelial progenitor and mesenchymal stem cell marker), integrin alpha 7 (ITGA7; a smooth, cardiac and skeletal muscle marker), as well as the C-X-C chemokine receptor type 4 (CXCR4; a lymphocyte, hematopoietic, and satellite cell marker)5. In basal conditions, satellite cells reside in a particular microenvironment that keeps them in a quiescent state6. Upon muscle damage, they become activated, proliferate, and undergo myogenesis7. However, contributing only to a minor fraction of the total number of muscle cells, their genome-wide analyses are particularly challenging, especially under physiological settings (&#60;1% of total cells).
Various methods for chromatin isolation from satellite cells have been described, which involve chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) or cleavage under targets and tagmentation (CUT&#38;Tag) experiments. Nevertheless, these two techniques present some significant limitations that remain unchallenged. Indeed, ChIP-seq requires a high amount of starting material to generate enough chromatin, a large proportion of which is lost during the sonication step. CUT&#38;Tag is more appropriate for low cell number, but generates more off-target cleavage sites than ChIP-seq due to the Tn5 transposase activity. In addition, since this enzyme has a high affinity for open-chromatin regions, the CUT&#38;Tag approach might be preferentially used for analyzing histone modifications or transcription factors associated with actively transcribed regions of the genome, instead of silenced heterochromatin8,9.
Presented here is a detailed protocol that allows the isolation of mouse limb muscle satellite cells by FACS for cleavage under targets and release using nuclease (CUT&#38;RUN)10,11 analysis. The various steps involve the mechanical disruption of tissue, cell sorting, and nuclei isolation. The method&#39;s efficiency, regarding the preparation of a viable cell suspension, was demonstrated by performing CUT&#38;RUN analysis for covalent histone modifications and transcription factors. The quality of isolated cells makes the described method particularly attractive for preparing chromatin that captures the native genomic occupancy state faithfully, and is likely to be suitable for capturing the chromosome conformation in combination with high-throughput sequencing at specific loci (4C-seq) or at genome-wide levels (Hi-C).

<table><tbody><tr><td>1.5 mL microtube</td><td>Eppendorf</td><td>2080422</td><td></td></tr><tr><td>2 mL microtube</td><td>Star Lab</td><td>S1620-2700</td><td></td></tr><tr><td>5 mL tubes</td><td>CORNING-FALCON</td><td>352063</td><td></td></tr><tr><td>50 mL tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>anti-AR</td><td>abcam</td><td>ab108341</td><td></td></tr><tr><td>anti-CD11b</td><td>eBioscience</td><td>25-0112-82</td><td></td></tr><tr><td>anti-CD31</td><td>eBioscience</td><td>12-0311-82</td><td></td></tr><tr><td>anti-CD34</td><td>eBioscience</td><td>48-0341-82</td><td></td></tr><tr><td>anti-CD45</td><td>eBioscience</td><td>12-0451-83</td><td></td></tr><tr><td>anti-CXCR4</td><td>eBioscience</td><td>17-9991-82</td><td></td></tr><tr><td>anti-DMD</td><td>abcam</td><td>ab15277</td><td></td></tr><tr><td>anti-H3K27ac</td><td>Active Motif</td><td>39133</td><td></td></tr><tr><td>anti-H3K4me2</td><td>Active Motif</td><td>39141</td><td></td></tr><tr><td>anti-ITGA7</td><td>MBL</td><td>k0046-4</td><td></td></tr><tr><td>anti-PAX7</td><td>DSHB</td><td>AB_528428</td><td></td></tr><tr><td>anti-TER119</td><td>BD Pharmingen &lt;sup&gt;TM&lt;/sup&gt;</td><td>553673</td><td></td></tr><tr><td>Beads</td><td>Polysciences</td><td>86057-3</td><td>BioMag&amp;reg;Plus Concanavalin A</td></tr><tr><td>Cell Strainer 100 &amp;micro;m</td><td>Corning&amp;reg;&amp;nbsp;</td><td>431752</td><td></td></tr><tr><td>Cell Strainer 40 &amp;micro;m</td><td>Corning&amp;reg;&amp;nbsp;</td><td>431750</td><td></td></tr><tr><td>Cell Strainer 70 &amp;micro;m</td><td>Corning&amp;reg;&amp;nbsp;</td><td>431751</td><td></td></tr><tr><td>Centrifuge 1</td><td>Eppendorf</td><td>521-0011</td><td>Centrifuge 5415 R</td></tr><tr><td>Centrifuge 2</td><td>Eppendorf</td><td>5805000010</td><td>Centrifuge 5804 R</td></tr><tr><td>Chamber Slide System&amp;nbsp;</td><td>ThermoFischer</td><td>171080</td><td>Syst&amp;egrave;me Nunc&amp;trade; Lab-Tek&amp;trade; Chamber Slide</td></tr><tr><td>Cleaning agent</td><td>Sigma&amp;nbsp;&amp;nbsp;</td><td>SLBQ7780V</td><td>RNaseZAPTM</td></tr><tr><td>Collagenase, type I&amp;nbsp;</td><td>Thermo Fisher</td><td>17100017</td><td>10 mg/mL</td></tr><tr><td>Dispase&amp;nbsp;</td><td>STEMCELL technologies</td><td>7913</td><td>5 U/mL</td></tr><tr><td>DynaMag&amp;trade;-2 Aimant</td><td>Invitrogen</td><td>12321D</td><td></td></tr><tr><td>Fixable Viability Stain</td><td>BD Biosciences</td><td>565388</td><td></td></tr><tr><td>Flow cytometer</td><td>BD FACSAria&amp;trade; Fusion Flow Cytometer</td><td>23-14816-01</td><td></td></tr><tr><td>Fluoromount G with DAPI</td><td>Invitrogen</td><td>00-4959-52</td><td></td></tr><tr><td>Genome browser&amp;nbsp;</td><td>IGV</td><td></td><td>http://software.broadinstitute.org/software/igv/</td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G9012</td><td></td></tr><tr><td>Hydrogel</td><td>Corning&amp;reg;&amp;nbsp;</td><td>354277</td><td>Matrigel hESC qualified matrix</td></tr><tr><td>Image processing software</td><td>Image J&amp;reg;</td><td></td><td>V 1.8.0</td></tr><tr><td>Laboratory film</td><td>Sigma-Aldrich</td><td>P7793-1EA</td><td>PARAFILM&amp;reg; M</td></tr><tr><td>Liberase LT</td><td>Roche</td><td>5401020001</td><td></td></tr><tr><td>Propyl gallate</td><td>Sigma-Aldrich</td><td>2370</td><td></td></tr><tr><td>Sequencer&amp;nbsp;</td><td>Illumina Hiseq 4000</td><td>SY-401-4001</td><td></td></tr><tr><td>Shaking water bath</td><td>Bioblock Scientific polytest 20</td><td>18724</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Cistrome Analysis in Mouse Muscle Stem Cells at JoVE.com

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

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).

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 ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.2K Views.  Université de Strasbourg. Al meer dan 20 jaar is ons labo geïnteresseerd in de pathofysiologische rol van nucleaire receptoren. Om hun in vivo functie te ontcijferen, hebben we een muismodel ontwikkeld dat de ruimtelijke en temporele controle van hun expressie mogelijk maakt. Tegelijkertijd ontwikkelden we geavanceerde technieken om hun moleculaire functie in de muizenweefsels te onderzoeken.Onze wetenschappelijke interesse heeft ons er onlangs toe gebracht om de signalering van secosteroïden en steroïderece...

Video: Auteur in de schijnwerpers: Cistrome-analyse in spierstamcellen van muizen 

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.

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 ...

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.

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.

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

Immunology and Infection

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Profiling Genomic Histone Marks in Mouse Myoblasts Using CUT&#38;Tag

Using Cleavage Under Targets and Tagmentation (CUT&#38;Tag) Assay in Mouse Myoblast Research

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&amp;Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&amp;Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&amp;Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&amp;Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

Author Spotlight: Navigating Challenges and Innovations in Muscle Stem Cell Studies

Researchers new to the epigenetic field will find CUT&amp;Tag a significantly easier alternative to ChIP assays. CUT&amp;Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&amp;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Neuroscience

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Biology

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

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

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