Tyssuelyser lt

ROS levels were quantified in liver homogenate using dihydrodichlorofluorescein diacetate prove (DCF-DA, Sigma-Aldrich, Madrid, Spain). Approximately 50 mg of liver was homogenized with Tyssuelyser LT (Qiagen, Hilden, Germany) for 1 min at 50 oscillations/s in 500 µL of RIPA lysis buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA). The liver homogenates were centrifuged at 1600× g for 10 min and the supernatant obtained was known as total liver homogenated and was stored at −80 °C for the assessment of protein levels and ROS. The liver homogenates were quantified by the standardized BCA method (Bio-RadProtein Assay; BioRad, Hercules, CA, USA). Briefly, 90 µL of liver homogenate (1:10) was dispensed into a 96 well black plate to which 10 mL of DCFDA was added to a final concentration of 10 µM and incubated for 30 min at room temperature to allow the cleavage of DCFDA by esterases and further conversion into the fluorescent product dichloro- fluorescein. The fluorescence was measured using a multimode plate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany) with excitation at 480 nm and emission at 530 nm. Results were normalized using total protein concentration (BCA protein assay, Thermo Fisher Scientific, Madrid, Spain) in each sample.

Total microbial DNA was extracted from faeces using the DNA stool mini kit (Qiagen, UK) by introducing three 1-min steps at 50 movements/s using TyssueLyser LT (Qiagen, UK) with 5-min incubation in ice between treatments as previously described by Candela et al. 2016 [59 (link)]. DNA recovery was evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies).

Approximately 20 mg of liver was homogenized with Tyssuelyser LT (Qiagen, Hilden, Germany) for 50 s in 300 µL lysis buffer (8 mmol/L NaH₂PO₄, 42 mmol/L Na₂HPO₄, 1% SDS, 0.1 mol/L NaCl, 0.1% NP40, 1 mmol/L NaF, 10 mmol/L sodium orthovanadate, 2 mmol/L PMSF, and 1% protease inhibitor cocktail 1 (Millipore Sigma, Darmstadt, Germany)). The protein extracts were quantified by the standardized BCA method (Bio-Rad Protein Assay; BioRad, Hercules, CA, USA). Protein extracts (20–25 µg) were electrophoretically separated on 10% SDS-PAGE and electroblotted to nitrocellulose membranes (Li-cor biosciences, NE, USA) [32 (link)]. Efficient protein transfer was monitored by Ponceau-S stain. Next, membranes were blocked (5% BSA) at room temperature and probed with specific primary antibodies (diluted 1:1000) overnight at 4º C in 1% BSA: total Akt (4685) (CST, Danvers, MA, USA), phospho-Akt (Ser473) (4060) (CST) and β-Actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA). Thereafter, infrared fluorescent secondary antibodies anti-rabbit 680, anti-rabbit 800 and anti-mouse 680 (LI-COR Biosciences, Lincoln, NE, USA; 926-32211, 926-68071 and 926-68070, respectively) were used for detection and quantified using ImageJ [33 (link)].