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

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

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).

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

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

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  

To begin, spray the sacrificed 10-week-old C57 black 6J male mouse thoroughly with 70%ethanol. Using forceps, remove the skin from the hindlimb and dissect all limb muscles surrounding the femur, tibia, and fibula. Place the harvested limb muscles into a two milliliter tube containing one milliliter of muscle isolation buffer.And using scissors, mince the harvested muscles. Transfer the minced muscle suspension into a 50 milliliter tube containing 18 milliliters of muscle isolation buffer. Close the tube tightly, seal it with laboratory film, and place it horizontally in a shaking water bath.After 30 minutes, add five milligrams of type I collagenase and maintain the tube for another 30 minutes. After pipetting the muscle suspension up and down 10 times, centrifuge and discard the supernatant, leaving five milliliters of the medium in the tube. Pipette the suspension onto the successive cell strainers and collect the flow-through into the 50 milliliter tube.Centrifuge the suspension and discard the supernatant until only 100 to 200 microliters remain in the tube. Resuspend the pellet in two milliliters of red blood cell lysis buffer and incubate on ice for three minutes. Centrifuge again and remove the supernatant from the tube using a 20 to 200 microliter pipette.Resuspend the pellet in 100 microliters of cold FACS buffer and place the tube on ice. After removing 10 microliters of cell suspension for the negative control, centrifuge the remaining 90 microliters and discard the supernatant before incubating the cells with 400 microliters of fixable viability stain diluted in serum-free DMEM for 15 minutes at room temperature. After centrifugation, add 100 microliters of FACS buffer and gently invert the tube thrice.Centrifuge again and add 100 microliters of primary antibody mixture into the tube. After 30 minutes of incubation in the dark, centrifuge and discard the supernatant. Then add 500 microliters of PBS to wash the cells.Centrifuge again and remove the supernatant before resuspending the pellet in 500 microliters of FACS buffer. Transfer the suspension to a five milliliter tube. Briefly vortex the cell suspension before processing it on a flow cytometer.Collect the selected cells in the five milliliter coated tube containing FACS buffer. 75%of the mononucleated cells identified based on their size and granularity were negative for the fixable viability stain marker leading to an average of 34.3%of live cells. About 3%of single living cells were negative for monocyte, endothelial, leukocyte, and erythroid specific markers.These negative cells were then selected according to their expression of CD34 and ITGA7 markers. A final gating for CXCR4 was performed to select putative satellite cells. And from the CD34-positive ITGA7-positive cells, 80%were found to be positive for CXCR4.

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234 Views.  Université de Strasbourg. To begin, spray the sacrificed 10-week-old C57 black 6J male mouse thoroughly with 70%ethanol. Using forceps, remove the skin from the hindlimb and dissect all limb muscles surrounding the femur, tibia, and fibula. Place the harvested limb muscles into a two milliliter tube containing one milliliter of muscle isolation buffer.And using scissors, mince the harvested muscles. Transfer the minced muscle suspension into a 50 milliliter tube containing 18 milliliters of muscle isolation buf...