Name: Preparation of Mouse Muscle Cell Suspension for Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting
Uploaded: 2023-07-07T14:20:00
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preparation of mouse muscle cell suspension for isolation of satellite cells by fluorescence-activated cell sorting

Isolatie van satellietcellen uit de spieren van muizenledematen en flowcytometrieanalyse

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).

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

Een efficiënt protocol voor CUT&amp;RUN-analyse van FACS-geïsoleerde muissatellietcellen

Since more than 20 years, our lab is interested in the pathophysiological role of nuclear receptors. To decipher their in vivo function, we engineered mouse model allowing the spatial and temporal control of their expression. In parallel, we developed cutting-edge techniques to investigate their molecular function in the mouse tissues.Our scientific interest recently led us to determine secosteroid and steroid receptor signaling in various tissues, including skeletal muscle. We have recently demonstrated that the androgen receptor in myofibers that differentiate cells of the skeletal muscle is instrumental in their contractile and metabolic functions. Our recent findings deliver a deeper understanding of skeletal muscle pathophysiological dynamics that is crucial to develop effective treatments for muscle-associated disorders.We developed this protocol to be able to perform cistrome analysis on muscle stem cells in vivo. All former protocols used in vitro modals, which completely dissociates the investigation from a whole organism context. In addition to being low-cost and time-efficient, this protocol provides an effective experimental setting to study transcription factor recruitment and chromatin landscape in skeletal muscle progenitor 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. Among many, our future aim would be to extend these investigations onto other oxosteroid receptors and explore their specific co-regulators in the various cell types involved in the regeneration process in skeletal muscle.

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Preparation of Mouse Muscle Cell Suspension for Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting  

Bereiding van spiercelsuspensie van muizen voor isolatie van satellietcellen door middel van fluorescentie-geactiveerde celsortering

Spuit om te beginnen de geofferde 10 weken oude C57 zwarte 6J mannelijke muis grondig in met 70% ethanol. Verwijder met een tang de huid van de achterpoot en ontleed alle ledemaatspieren rond het dijbeen, het scheenbeen en het kuitbeen. Plaats de geoogste ledemaatspieren in een buis van twee milliliter met één milliliter spierisolatiebuffer.En hak met een schaar de geoogste spieren fijn. Breng de gehakte spiersuspensie over in een buis van 50 milliliter met 18 milliliter spierisolatiebuffer. Sluit de buis goed af, sluit hem af met laboratoriumfolie en plaats hem horizontaal in een schudwaterbad.Voeg na 30 minuten vijf milligram collagenase type I toe en houd de tube nog eens 30 minuten vast. Nadat u de spiersuspensie 10 keer op en neer hebt gepipetteerd, centrifugeert u en gooit u het supernatant weg, waarbij u vijf milliliter van het medium in de buis achterlaat. Pipetteer de suspensie op de opeenvolgende celzeven en vang de doorstroming op in de buis van 50 milliliter.Centrifugeer de suspensie en gooi het supernatans weg totdat er nog maar 100 tot 200 microliter in de buis zit. Resuspendeer de pellet in twee milliliter lysebuffer voor rode bloedcellen en incubeer gedurende drie minuten op ijs. Centrifugeer opnieuw en verwijder het supernatans uit de buis met behulp van een pipet van 20 tot 200 microliter.Resuspendeer de pellet in 100 microliter koude FACS-buffer en plaats de buis op ijs. Na het verwijderen van 10 microliter celsuspensie voor de negatieve controle, centrifugeert u de resterende 90 microliter en gooit u het supernatans weg voordat u de cellen incubeert met 400 microliter fixeerbare levensvatbaarheidskleuring verdund in serumvrije DMEM gedurende 15 minuten bij kamertemperatuur. Voeg na het centrifugeren 100 microliter FACS-buffer toe en keer de buis voorzichtig driemaal om.Centrifugeer opnieuw en voeg 100 microliter primair antilichaammengsel toe aan de buis. Na 30 minuten incubatie in het donker, centrifugeer en gooi het supernatant weg. Voeg vervolgens 500 microliter PBS toe om de cellen te wassen.Centrifugeer opnieuw en verwijder het supernatans voordat u de pellet opnieuw suspendeert in 500 microliter FACS-buffer. Breng de suspensie over in een buis van vijf milliliter. Draai de celsuspensie kort in een draaikolk voordat u deze op een flowcytometer verwerkt.Verzamel de geselecteerde cellen in de vijf milliliter gecoate buis met FACS-buffer. 75% van de eenkernige cellen die werden geïdentificeerd op basis van hun grootte en granulariteit waren negatief voor de fixeerbare levensvatbaarheidsvlekmarker, wat leidde tot een gemiddelde van 34,3% van de levende cellen. Ongeveer 3% van de afzonderlijke levende cellen was negatief voor monocyten, endotheel, leukocyten en erytroïde specifieke markers.Deze negatieve cellen werden vervolgens geselecteerd op basis van hun expressie van CD34- en ITGA7-markers. Een laatste gating voor CXCR4 werd uitgevoerd om vermeende satellietcellen te selecteren. En van de CD34-positieve ITGA7-positieve cellen bleek 80% positief te zijn voor CXCR4.

Research

JoVE Journal

Developmental Biology

Preparation of Mouse Muscle Cell Suspension for Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting

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 ...