Ultra turrax

Neutrophil-differentiated HL-60 cells and/or cardiomyocytes were homogenized in lysis buffer (in mM): 50 Tris; 30 NaCl; 2 EDTA, 0.1% SDS, and Protein Inhibitor Cocktail (Complete Mini EDTA-free, Roche, USA) using T 10 basic Ultra-Turrax^® in speed scale 4 (approx. 22,000 rpm). Each sample was then centrifuged at 13,200 rpm for 15 min at 4 °C; the pellet was discarded, and the supernatant was stored for future analysis.
The total protein was quantified by the bicinchonic acid method (BCA), using the commercial BCA ™ kit (Thermo Scientific Pierce ™, Rockford, IL, USA), using BSA as standard. Briefly, the assay was performed following the manufacturer’s instructions for the microplate reading protocol (Thermo Scientific Pierce BCA Protein Assay Kit, Rockford, IL, USA, 2007: 1–7), where 25 μL of the sample was mixed with 200 mL of the working reagent that was prepared at the time, and the mixture was incubated for 30 min at 37 °C. An 8-point calibration curve was also prepared, covering albumin concentrations ranging from 25 to 2000 μg/mL, in addition to a reagent blank as recommended by the manufacturer. The ThermoScientific™ Multiskan™ microplate spectrophotometer (Thermo Scientific, Rockford, IL, USA) was used to perform agitation, incubation, and reading of the absorbances at 562 nm.

Cytokine bead assay was performed with the FlowCytomix System (eBioscience, Frankfurt, Germany) according to the manufacturer’s manual. One half of a brain per mouse was lysed in 50 mM Tris pH 7.4 with cOmplete Ultra Tablet (Roche, Basel, Switzerland) with an Ultra-Turrax (5 section at 17500 rpm) on ice. Lysates were centrifuged at 10000 x g, 4°C for 30 min. Protein levels in supernatants were measured by BCA Protein Assay (Pierce, Rockford, USA).

Samples were prepared as previously described^{25 (link)}. A volume of 200 mg of liver tissue was homogenized in 2.5 mL of 10 mM HEPES with a pH of 7.4 supplemented with 0.25 M sucrose, 5 mM EDTA and a protease inhibitor cocktail (Roche) for 3 × 10 s in a 6 mm Ultra Turrax homogenizer. The homogenate was incubated on ice for 30 min and centrifuged at 400 × g, 4 °C, for 10 min. The supernatant was centrifuged at 3000 × g for 15 min, the 3000 × g pellet was homogenized in 1 mL of 2 M NaCl in10 mM HEPES. Another centrifugation at 3000 × g for 15 min was performed. The pellet was again homogenized in 0.1 M sodium carbonate and incubated for 1 h while agitating. After centrifugation at 16,000 × g for 1 h the pellet was homogenized in 1 mL of 10 mM HEPES with 4 M urea and incubated on ice for 30 min. The homogenate was centrifuged at 16,000 × g. The final pellet was washed with 10 mM HEPES and re-suspended in 125 µL of 25 mM ammonium bicarbonate with 2% SDS. Protein samples were subsequently used for immunoblot analysis.