Cells were cultured on 100-mm plates and washed twice with ice-cold PBS. Harvested cells were homogenized in buffer A (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM phenylmethylsulphonyl fluoride, 20 mM HEPES (pH 7.5) and protease inhibitor cocktail (Roche)) using a Teflon pestle (Thomas Scientific) and then centrifuged at 1,000g at 4 °C for 10 min. Pellets were used as nuclear fractions while post-nuclear supernatant fractions were centrifuged at 10,000g at 4 °C for 10 min to obtain mitochondria. The pellets containing mitochondria were washed with buffer A and resuspended in TXIP-1 buffer (1% Triton X-100, 150 mM NaCl, 0.5 mM EDTA and 50 mM Tris-HCl (pH 7.4)) containing a protease inhibitor cocktail. The post-mitochondrial supernatant was centrifuged for 30 min at 100,000g to obtain cytosolic protein fractions.
Teflon pestle
Manufactured by Thomas Scientific
Sourced in United States
The Teflon pestle is a laboratory tool used for grinding and mixing solid samples in a mortar. It is made of Teflon, a durable and non-stick material, which allows for efficient sample processing and easy cleanup.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thomas Scientific (1 mentions)
Saline solution, by Merck Group (1 mentions)
Phosphate buffer, by Merck Group (1 mentions)
Ultra turrax, by IKA Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Cholesterol quantification kit, by Abcam (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
4 protocols using teflon pestle
1
Subcellular Fractionation Protocol
2
Mitochondrial Extraction and Enzymatic Assays
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TK2 and littermate controls were sacrificed at early stage disease (p15–18), and ∼0.3 g tissue were immediately homogenized on ice in an isotonic and viscous mitochondrial extraction buffer (70 mM sucrose, 210 mM mannitol, 5 mM HEPES, and 1 mM EDTA, pH 7.2) using 15 strokes in a glass Dounce tissue grinder with a Teflon® pestle (Thomas Scientific). Aliquots of total lysate were saved for mtDNA and western blot analysis. To enrich for mitochondria, the remaining homogenate was centrifuged at 900×g for 10 min at 4 °C. Supernatant was transferred and re-centrifuged at 900×g for 10 min at 4 °C, followed by a final transfer and re-centrifugation at 4000×g for 10 min at 4 °C. Finally, supernatant was removed and discarded, and the resulting mitochondrial-enriched pellet was re-suspended in mitochondrial assay solution (70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and 0.2% fatty acid-free BSA, pH 7.4) to a concentration of approximately 10 µg/µl. The resulting mitochondrial enrichment sample was used to assess respiratory chain and CPT1 enzymatic activities, and for western blot analysis.
3
Retinal Tissue Isolation and Preparation
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Immediately after sacrifice, eyes were enucleated and transferred to a saline solution with a pH of 7.4 (Sigma Aldrich). Retinas were rapidly dissected by making a small incision with a scalpel blade at 1 mm behind the limbus. The incision was then extended 360° around the globe using fine ophthalmic scissors. Anterior segment structures (cornea, iris, and lens) were removed and the retina was separated from the RPE-choroidal complex. Retinas were homogenized using an Ultra-Turrax (Ika Werke GmbH & Co. KG, Staufen, Germany) to prepare them for posterior reverse transcription polymerase chain reaction (RT-PCR) analyses. The RPE-choroid samples were also homogenized, but with a Teflon pestle (Thomas Scientific, Swedesboro, NJ, USA) in 75 µL of phosphate buffer (Sigma Aldrich). The RPE-choroid samples were then centrifuged (13,000 rpm) for 20 min at 4 °C. The resulting supernatant was collected and protein concentration was determined using a slightly modified Bradford assay (Bio-Rad, Hercules, CA, USA) [28 (link)].
4
Maternal-Fetal Lipid Profiling
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