550 sonic dismembrator

Four batches of magnetite obtained from ferric chloride hexahydrate and ferrous chloride tetrahydrate (at a molar ratio 2:1, weighing 520 mg and 192 mg, respectively dissolved in 6.2 mL of doubly deionized water) by alkaline precipitation with aqueous sodium hydroxide (a 3.8 mL of 2N sodium hydroxide) were magnetically separated and washed twice with 5 mL of water^{26 (link), 27 (link)}. All four batches (maximal theoretical amount of 890 mg) of magnetite were combined and dispersed in 11 mL of 1.2% (w/v) alginate solution. The magnetite suspended in alginate solution was heated in a water bath (90°C) for 20 min with a periodic mixing. Then the suspension was cooled down to a room temperature and sonicated for 5 min using an acoustic probe (0.5 inch diameter; 5 inch length) operated at 35-40% power corresponding to 190-220 Watts (550 Sonic Dismembrator, Fisher Scientific, USA) keeping the tube chilled on ice-water. The heating and sonication cycles were repeated twice resulting in a stable ferrofluid with a final magnetite concentration of ~8% (w/v). Particle size measurements were determined by Photon Correlation Spectroscopy (PCS) using a Delsa^TM Nano C particle size analyzer equipped with two laser diodes (658 nm, 30 mW, Beckman-Coulter, CA, USA).

Cells were lysed and total protein was extracted with sonication (10 s, Setting 3, Fisher 550 Sonic dismembrator) in extraction buffer (1% v/v NP-40 in Tris-buffered saline (TBS; 50 mm Tris-HCl, pH 7.5, 150 mm NaCl)) with 0.5 mM PMSF, PIC1, PIC2, 500nM Thiamet-G, 10 mM β-hexosaminidase inhibitor, 5 mM NaF, and 5 mM β-glycerophosphate). Cellular debris were pelleted at 18,000 ×g (30 min at 4 °C). Protein concentration was estimated using the Pierce 600nm Protein Assay Reagent (Pierce Biotechnology). Typically, 1 μg of antibody per 500 μg of protein extract was used for immunoprecipitations. Protein A/G magnetic beads (GE Healthcare) were used to capture the antibody:protein complex. Immunoprecipitates were washed with TBS + 1% v/v NP-40 (3x) to remove non-specifically bound proteins, and then resuspended in 1x LDS buffer with 50 mM DTT, and heated at 85°C for 10 min.

Brain samples were homogenized in ice-cold isolation buffer (0.32 M sucrose, 20 mM HEPES, 2 mM EDTA, 2 mM EGTA, 0.1 M GlcNAc, pH 7.4 with 4 μg/ml leupeptin, 4 μg/ml pepstatin A, 5 μg/ml aprotinin, 0.2 mM PMSF) using a Wheaton glass homogenizer. One aliquot was set on ice and sonicated 2x 10s on 20% power using a 550 Sonic Dismembrator (Fisher Scientific, Rockford, IL) and centrifuged at 4°C for 10 minutes at 1000 x g to obtain homogenates. A different aliquot was subjected to subcellular fractionation. Samples were first centrifuged at 4°C for 10 minutes at 1000 x g, the supernatant (crude cytosolic fraction) was transferred into a new tube and centrifuged again at 4°C for 10 minutes at 16,090 x g. The resulting supernatant represents the cytosolic fraction. Protein estimation was performed using Pierce BCA Protein Assay (Thermo Scientific, Rockford, IL).

We isolated intact chromatin from primary hepatocytes (~5×10⁶ cells) by using ChIP-IT Express Kit (Active Motif) following the manufacturer’s instructions; cells were sonicated using the 550 Sonic Dismembrator (Fisher Scientific). Chromatin immunoprecipitation was followed by qPCR using using GoTaq® qPCR Master Mix (Promega). Multiple overlapping pairs of primers were designed using Primer3 to cover hepatic Gck promoter from −1447 to +52 from the start point. P5, P20, P21 and P22 sequences were as follow: P5 (forward) 5′-ATCCGCTCCGTTTGTCTCT-3′ and (reverse) 5′-ATCTCCTGGGCAAGTCACAG-3′ (−1187 to −1040); P20 (forward) 5′-GAAGGGGGCATGTGAGTG-3′ and (reverse) 5′-AAAGAACCACGTGGGATCAG-3′ (−219 to −77); P21 (forward) 5′-GTGTTCAGAGAACATGGTAGCC-3′ and (reverse) 5′-TCTGAGAGGTGGCTCCTAAAA-3′ (−154 to −9); P22 (forward) 5′-ATCCCACGTGGTTCTTTGTC-3′ and (reverse) 5′-ACTGTCTGGCTGAGTGTTGC-3′ (−93 to +52). Other primer sequences are available upon request. Fold enrichment was calculated by a modified ΔC(t) method and normalized to DNA immunoprecipitated with negative IgG control (Sigma) antibody according to the formula (Δ(C(t)IP – C(t)input)100.