Isolation of Skeletal Muscle Nuclei

Abstract

A method employing aqueous media for isolation of nuclei from rat skeletal muscle is described. The technique involves (a) mincing and then homogenizing in a 0.32 M sucrose-salt solution with a Potter-Elvehjem type homogenizer using a Delrin (an acetal resin) pestle and a carefully controlled, relatively large pestle-to-glass clearance, (b) filtering through fiberglass and stainless steel screens of predetermined mesh size to remove myofibrils and connective tissue, and (c) centrifuging in a 2.15 M sucrose-salt solution containing 0.7 m_u ATP. Electron and phase-contrast microscopic observations show that the nuclei are intact, unencumbered by cytoplasmic tags, and possess well preserved distinct nucleoli, nucleoplasm, and nuclear membranes. Cytoplasmic contamination is minimal and mainly mitochondrial. Chemical assays of the nuclear flacfion show that the DNA/protein and RNA/ DNA ratios are comparable to those obtained in other tissues. These ratios, as well as the low specific activity obtained for cytochrome c oxidase and the virtual absence of myofibrillar ATPase, indicate a high degree of purity with minimal mitochondrial and myofibrillar contamination. The steps comprising the technique and the reasons for their selection are discussed.

Fatty acids acutely enhance insulin-induced oxidative stress and cause insulin resistance by increasing mitochondrial reactive oxygen species (ROS) generation and nuclear factor-κB inhibitor (IκB)–nuclear factor-κB (NFκB) activation in rat muscle, in the absence of mitochondrial dysfunction

“…Electrophoretic mobility shift assay Nuclei from rat muscle were prepared with a modified version of previously described methods [21]. Tissue samples were homogenised in a buffered sucrose-salt solution (0.2 mmol/l K 2 HPO 4 , 0.6 mmol/l KH 2 PO 4 , 0.32 mol/l sucrose, 1 mmol/l MgCl 2 , pH06.8) with protease inhibitors (Sigma), nuclei were collected by centrifugation (800×g, 5 min, 4°C) and washed with the same procedure.…”

Suppression of the GLUT4 adaptive response to exercise in fructose-fed rats

“…After discarding of the supernatant, the pellet was resuspended in 300 l of cold homogenization buffer, and after centrifugation the final pellet was resuspended in 200 l of nuclei suspension buffer (18.5% glycerol, 11.1 mM MgCl2, 14.8 mM HEPES, 0.33 mM NaCl, 75 mM KCl and 1ϫ RCPI). Protein concentration of the nuclei suspension was first determined using the Bradford assay and the DNA concentration estimated by multiplying the protein concentration by a factor of 0.17 (9).…”

“…ChIP assays were performed on triceps muscle for the assessment of 1) the level of acetylation of histone H3 on Lys 9 and Lys 14 within a 350-bp DNA containing the MEF2 binding site on the Glut4 gene promoter and 2) the binding of MEF2A to its binding site on the Glut4 gene promoter. Approximately 100 mg frozen triceps muscle was crushed to a fine power in liquid nitrogen and cross-linked using 1% formaldehyde in phosphate-buffered saline (PBS), pH 7.40, for 10 min at room temperature.…”

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle <em>In Situ</em> Using the TUNEL Method

“…The comet assay is an alternative cell-based method that requires extracting preserved nuclei from cells 4,20,28,36 . Although the comet assay works well on cultured isolated cells, it is much more difficult to prepare intact nuclei from skeletal muscle tissue 8,21 . As with Southern blot, the comet assay does not provide cell-type-specific information from a whole muscle tissue homogenate.…”