Name: Preparation of Mouse Muscle Cell Suspension for Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting
Uploaded: 2023-07-07T14:20:00
Description: Watch this Scientific Journal Video about Preparation of Mouse Muscle Cell Suspension for Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting at JoVE.com

preparation of mouse muscle cell suspension for isolation of satellite cells by fluorescence-activated cell sorting

Fare Uzuv Kaslarından Uydu Hücre İzolasyonu ve Akış Sitometrisi Analizi

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).

Yazar Spotlight: Fare Kas Kök Hücrelerinde Sistrom Analizi 

FACS ile İzole Edilmiş Fare Uydu Hücrelerinin CUT&amp;RUN Analizi için Etkili Bir Protokol

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.

Watch this Scientific Journal Video about Preparation of Mouse Muscle Cell Suspension for Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting at JoVE.com

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

Floresanla aktive edilen hücre ayırma ile uydu hücrelerinin izolasyonu için fare kas hücresi süspansiyonunun hazırlanması

Başlamak için, kurban edilen 10 haftalık C57 siyah 6J erkek fareye %70 etanol iyice püskürtün. Forseps kullanarak cildi arka bacaktan çıkarın ve femur, tibia ve fibulayı çevreleyen tüm uzuv kaslarını inceleyin. Hasat edilen uzuv kaslarını, bir mililitre kas izolasyon tamponu içeren iki mililitrelik bir tüpe yerleştirin.Ve makas kullanarak, hasat edilen kasları kıyın. Kıyılmış kas süspansiyonunu 18 mililitre kas izolasyon tamponu içeren 50 mililitrelik bir tüpe aktarın. Tüpü sıkıca kapatın, laboratuvar filmi ile kapatın ve çalkalanan bir su banyosuna yatay olarak yerleştirin.30 dakika sonra, beş miligram tip I kollajenaz ekleyin ve tüpü 30 dakika daha koruyun. Kas süspansiyonunu 10 kez yukarı ve aşağı pipetledikten sonra, süpernatanı santrifüjleyin ve atın, tüpte beş mililitre ortam bırakın. Süspansiyonu ardışık hücre süzgeçlerine pipetleyin ve akışı 50 mililitrelik tüpe toplayın.Süspansiyonu santrifüjleyin ve tüpte sadece 100 ila 200 mikrolitre kalana kadar süpernatanı atın. Peletleri iki mililitre kırmızı kan hücresi lizis tamponunda yeniden süspanse edin ve üç dakika boyunca buz üzerinde inkübe edin. Tekrar santrifüjleyin ve 20 ila 200 mikrolitrelik bir pipet kullanarak süpernatanı tüpten çıkarın.Peletleri 100 mikrolitre soğuk FACS tamponunda tekrar süspanse edin ve tüpü buzun üzerine yerleştirin. Negatif kontrol için 10 mikrolitre hücre süspansiyonunu çıkardıktan sonra, kalan 90 mikrolitreyi santrifüjleyin ve hücreleri oda sıcaklığında 15 dakika boyunca serumsuz DMEM içinde seyreltilmiş 400 mikrolitre sabitlenebilir canlılık lekesi ile inkübe etmeden önce süpernatanı atın. Santrifüjlemeden sonra, 100 mikrolitre FACS tamponu ekleyin ve tüpü üç kez hafifçe ters çevirin.Tekrar santrifüjleyin ve tüpe 100 mikrolitre primer antikor karışımı ekleyin. Karanlıkta 30 dakika inkübasyondan sonra, süpernatanı santrifüjleyin ve atın. Sonra hücreleri yıkamak için 500 mikrolitre PBS ekleyin.Tekrar santrifüjleyin ve peleti 500 mikrolitre FACS tamponunda yeniden süspanse etmeden önce süpernatanı çıkarın. Süspansiyonu beş mililitrelik bir tüpe aktarın. Bir akış sitometresinde işlemeden önce hücre süspansiyonunu kısaca vorteksleyin.Seçilen hücreleri, FACS tamponu içeren beş mililitre kaplı tüpte toplayın. Boyutlarına ve tanecikliliklerine göre tanımlanan tek çekirdekli hücrelerin %75'i, canlı hücrelerin ortalama %34.3'üne yol açan sabitlenebilir canlılık leke işaretleyicisi için negatifti. Tek canlı hücrelerin yaklaşık% 3'ü monosit, endotelyal, lökosit ve eritroid spesifik belirteçler için negatifti.Bu negatif hücreler daha sonra CD34 ve ITGA7 belirteçlerinin ekspresyonlarına göre seçildi. Varsayılan uydu hücrelerini seçmek için CXCR4 için son bir geçit yapıldı. CD34 pozitif ITGA7 pozitif hücrelerin %80'inin CXCR4 pozitif olduğu bulundu.

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 ...