Application protocol

Dissociation of skeletal muscle tissue and isolation of functional satellite cells

This application protocol is based on a scientific poster that can be accessed under "Resources" in the Protocol tab.

In this application protocol, we describe an easy and fast method for the dissociation of skeletal muscle tissue from mouse and a subsequent isolation method that generates a highly pure population of satellite cells and minimizes bias in downstream applications caused by contamination with non-target cells.

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Protocol

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The following instructions generate a high-purity subfraction of functional and culturable satellite cells.

1. Dissociation of skeletal muscle tissue

Use the Skeletal Muscle Dissociation Kit to generate a single-cell suspension by combining mechanical dissociation with the gentleMACS™ Dissociator and enzymatic degradation of the extracellular matrix that maintains the structural integrity of the tissue. Follow the protocol of the kit data sheet.

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Skeletal Muscle Dissociation Kit, mouse and rat

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Dissociation of solid skeletal muscle tissue generates single-cell suspensions with high viability. The Skeletal Muscle Dissociation Kit combines enzymatic and mechanical treatment with the gentleMACS Dissociator to generate a single-cell suspension with low amounts of debris (left) and cell viabilities above 90% (right).

2. Isolation of satellite cells

Isolate functional satellite cells by depleting non-target cells with the Satellite Cell Isolation Kit. The kit contains a cocktail of monoclonal antibodies conjugated with MACS® MicroBeads the magnetically label non-target cells. These are retained within the MACS Column, while the unlabeled satellite cells pass through during elution. Follow the protocol of the kit data sheet.

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Satellite Cell Isolation Kit, mouse

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Depletion of non-target cells from a single-cell suspension of skeletal muscle tissue generates a high-purity subfraction of satellite cells. Cells expressing lineage markers (CD31, CD45, CD11b, Sca-1) were depleted from dissociated skeletal muscle tissue of mouse, leaving functional satellite cells that can be optionally purified further with a positive selection step based on integrin-alpha-7 expression.

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Characterization of isolated satellite cells. A and B: Isolated satellite cells give rise to myoblast cultures without pre-plating or selection media. C: Efficient in vitro expansion and differentiation into myotubes.

Resources

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Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec B.V. & Co. KG and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec B.V. & Co. KG and/or its affiliates. All rights reserved.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

Reagent and instrument requirements

General reagents and instrument requirements

PBE buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091‑222). Keep buffer cold (2−8 °C). Always use freshly prepared buffer. Do not use autoMACS Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as mouse or rat serum albumin, mouse or rat serum, or FBS. Buffers or media containing Ca²⁺ or Mg²⁺ are not recommended for use.

For skeletal muscle dissociation

Skeletal Muscle Dissociation Kit, mouse and rat (# 130-098-305)
Cell culture medium without fetal bovine serum (FBS), e.g., DMEM
gentleMACS™ Dissociator (# 130-093-235), gentleMACS OctoDissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
gentleMACS C Tubes (# 130-093-237, # 130-096-334)
MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubator at 37 °C
MACS SmartStrainers (70 μm) (# 130-098-462)
(Optional) Red Blood Cell Lysis Solution (10×) (# 130-094-183)
(Optional) ART. 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes

For isolation of satellite cells

Satellite Cell Isolation Kit, mouse (# 130-104-268)
PBS buffer
Pre-Separation Filters, 70 μm (# 130-095-823) to remove cell clumps
(Optional) Basic FGF/FGF2, e.g., Human FGF-2, premium grade (# 130-093-840) for culture of satellite cells
(Optional) Inside Stain Kit (# 130-090-477) to detect intracellular markers, such as Pax7
(Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometric exclusion of dead cells without fixation
(Optional) Anti-Integrin α-7 MicroBeads, mouse (# 130-104-261) to further increase the purity of isolated satellite cells, e.g., for direct molecular analysis, such as mRNA expression profiling.
MACS Columns and MACS Separators: For optimal purity and recovery use an LS Column. Depletion can also be performed using the autoMACS Pro Separator.

Column	Max. number of labeled cells	Max. number of total cells	Separator
LS	2×10⁷	4×10⁷	MidiMACS™, QuadroMACS™, VarioMACS, SuperMACS II
autoMACS	5×10⁷	1×10⁸	autoMACS Pro

▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.

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Application protocol

Dissociation of skeletal muscle tissue and isolation of functional satellite cells

This application protocol is based on a scientific poster that can be accessed under "Resources" in the Protocol tab.

Protocol

Materials