Around 5-mm fragments of spinal cord tissue centered on the injury site or corresponding site in sham animals were collected from sham mice, and at 2 or 24 h after and homogenized in ice-cold buffered solution containing 0.32 M sucrose, 10 mM Hepes with the addition of protease and phosphatase inhibitors24 (link). Homogenates were centrifuged at 800 × g for 10 min at 4 °C to pellet down the nuclei. Supernatants were sequentially centrifuged at 20,000 × g for 20 min at 4 °C to pellet the heavy membrane/crude lysosomal fractions and at 100,000 × g for 1 h at 4 °C to pellet light membrane fractions. Both supernatant and suspended pellet fractions were recentrifuged to minimize cross contamination from the different subcellular fractions. All pellets were resuspended in homogenization buffer. Protein concentration was estimated using BCA reagent; samples were analyzed by Western Blot.
For isolation of purified lysosome samples, after the 5 mm segment of the spinal cord was extracted, the lysosome enrichment kit (Thermo Scientific, Cat.) was used according to the manufacturer’s instructions to obtain a purified form of lysosome for downstream testing with Western Blot analysis and MASPEC lipidomic.
For isolation of purified lysosome samples, after the 5 mm segment of the spinal cord was extracted, the lysosome enrichment kit (Thermo Scientific, Cat.) was used according to the manufacturer’s instructions to obtain a purified form of lysosome for downstream testing with Western Blot analysis and MASPEC lipidomic.
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