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)