A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue

BMC Research Notes, Sep 2012

Background We describe a method for subcellular fractionation of mouse skeletal muscle, myoblast and myotubes to obtain relatively pure fractions of nuclear, cytosolic and mitochondrial compartments. Fractionation allows the analysis of a protein of interest (or other cellular component) based on its subcellular compartmental distribution and can also generate molecular information about the state of a cell and/or tissue and how the distribution of a protein may differ between different cellular compartments, tissues or cell types, in response to treatments or ageing. Findings The described method was specifically developed for skeletal muscle and proliferating/differentiated muscle cells. The purity of the different fractions, representing the cytoplasmic, mitochondrial and nuclear subcellular compartments was validated by western blot analysis of “house-keeper” marker proteins specific for each cellular compartment. Conclusion This low cost method allowed the mitochondrial, cytoplasmic and nuclear subcellular compartments from the same starting muscle samples to be rapidly and simultaneously isolated with good purity and without the use of an ultracentrifuge. This method permits samples to be frozen at −80°C for future analysis and/or additional processing at a later date.

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A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue

BMC Research Notes A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue Ivan Dimauro 1 Timothy Pearson 0 Daniela Caporossi 1 Malcolm J Jackson 0 0 Department of Musculoskeletal Biology, Institute of Ageing & Chronic Disease, University of Liverpool , Daulby Street, Liverpool L69 3GA , United Kingdom 1 Department of Health Sciences, University of Rome “Foro Italico” , Piazza Lauro De Bosis 15, 00194, Rome , Italy Background: We describe a method for subcellular fractionation of mouse skeletal muscle, myoblast and myotubes to obtain relatively pure fractions of nuclear, cytosolic and mitochondrial compartments. Fractionation allows the analysis of a protein of interest (or other cellular component) based on its subcellular compartmental distribution and can also generate molecular information about the state of a cell and/or tissue and how the distribution of a protein may differ between different cellular compartments, tissues or cell types, in response to treatments or ageing. Findings: The described method was specifically developed for skeletal muscle and proliferating/differentiated muscle cells. The purity of the different fractions, representing the cytoplasmic, mitochondrial and nuclear subcellular compartments was validated by western blot analysis of “house-keeper” marker proteins specific for each cellular compartment. Conclusion: This low cost method allowed the mitochondrial, cytoplasmic and nuclear subcellular compartments from the same starting muscle samples to be rapidly and simultaneously isolated with good purity and without the use of an ultracentrifuge. This method permits samples to be frozen at −80°C for future analysis and/or additional processing at a later date. Skeletal muscle; Subcellular fractionation; Western blotting Findings Background Isolation of nuclear, cytosolic and mitochondrial fractions of reasonable purity from mammalian tissues and cells has generated great interest as it has the advantage of allowing different cellular proteins and organelles to be studied and characterised. Subcellular fractionation is universally used for various cell types and tissues for sample preparation and prior to subsequent ~ omics analysis [ 1-5 ]. Generic fractionation protocols exist that can purify specific subcellular compartments and organelles, but in general they are not tailored for use with skeletal muscle and may require large amounts of starting material, time, or special reagents whilst potentially yielding fewer fractions from the same starting sample etc. [ 3,5-9 ]. The protocol described has been optimized for use with primary skeletal muscle tissue (e.g. mouse anterior tibialis (AT) muscle) and both proliferating and differentiated C2C12 cells to isolate subcellular fractions of nuclei, cytosol, and mitochondria from a single starting sample, thereby reducing the quantity of starting material, cost and total time needed for sample preparation. The protocol works well for skeletal muscle tissue and cells and could be used as a starting point for the fractionation of other non-muscle samples although changes to buffer volumes; homogenization duration/intensity etc. may be required. The purity of the fractions obtained was assessed by immunoblotting for specific protein markers: histone H3 (nuclei), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, cytosol), and cytochrome oxidase IV (CoxIV, mitochondria). Cell culture and animals The C2C12 mouse skeletal myoblast cell line was obtained from the American Type Culture Collection (CRL-1772). C2C12 myoblasts were maintained in DMEM (Sigma Aldrich, Poole, UK) supplemented with 1% L-glutamine (Lonza, Cologne, Germany), 10% FBS (Biosera, Sussex, UK) and 1% penicillin and streptomycin (Sigma) under an atmosphere of 5% CO2 in humidified air at 37°C. To induce myogenic differentiation, the growth medium was changed to differentiation medium (DMEM supplemented with 2% horse serum (Sigma) and 1% antibiotics) after myoblasts had reached 90% confluence in a T75 cm2 flask. Myoblast cells were either harvested at 90% confluence or allowed to mature to myotubes for 7 days and then harvested (see below). Adult mice (C57BL/6) were euthanized by overdose with anesthetic (ketamine hydrochloride and medatomidine hydrochloride) administered by intraperitoneal injection. Anterior tibialis (AT) muscles, approximately 50 mg wet weight, were rapidly removed and used fresh to prepare fractions. Experiments were performed in accordance with UK Home Office Guidelines under the UK Animals (Scientific Procedures) Act 1986 and received ethical approval from the University of Liverpool Animal Welfare Committee. Subcellular fractionation Fresh AT tissue and scraped cells were washed with cold PBS, cells were pelleted by centrifugation at 200 g for 7 minutes whereas tissues were placed in a pre-chilled glass Petri dish and minced on ice using sharp scissors. All samples were resuspended in 300-500 μl of STM buff (...truncated)


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Ivan Dimauro, Timothy Pearson, Daniela Caporossi, Malcolm J Jackson. A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue, BMC Research Notes, 2012, pp. 513, Volume 5, Issue 1, DOI: 10.1186/1756-0500-5-513