Pt1300d

At 3 and 7 days after MI, the hearts of unoperated control, Sham, MI and MI + ABR mice were harvested and washed with PBS containing a protease inhibitor cocktail. Next, the cardiac muscle below the ligature point of the left ventricle (LV) was removed, rapidly frozen with liquid nitrogen and preserved at −80 °C. RNA and proteins were extracted using TRIzol Reagent according to the manufacturer’s protocol. Briefly, LV tissue was homogenized in TRIzol Reagent using a Polytron PT 1300 D homogenizer (Kinematica, Lucerne, Switzerland), incubated with chloroform and centrifuged. The resulting upper aqueous phase was used for RNA precipitation, and the lower organic phase was used for protein precipitation. The protein pellet was solubilized in a lysis buffer containing 8 M urea, 1% sodium deoxycholate (DOC), 100 mM Tris-HCl (pH 7.5) and a protease inhibitor cocktail. Protein concentrations were determined by the BCA Protein Assay Kit. The purity and concentration of RNA was determined with a NanoDrop Lite spectrophotometer (Thermo Scientific). Only the RNA samples with 260/280 absorption ratio between 1.8 and 2.1 were used for further analysis.

The antifouling test was performed using an oily saline solution. The 3.5 wt % NaCl solution was spiked with 1000 ppm crude oil (ONTA, Midland, TX, USA). To obtain a homogeneous oily saline solution, the solution was mixed at 16,000 rpm for 30 min using the homogenizer (PT1300D, Kinematica Inc., Bohemia, NY, USA). The oil droplet size in the resulting solution was ~5 µm in a range of 1.3–9 µm and the oil volume fraction was ~0.0014 vol%, with no change in droplet size over 48 h. Nevertheless, the oil suspension was prepared immediately before starting the experiment. Permeate weight was recorded every 10 min.
The antiwetting properties of the membrane were investigated by challenging the membrane with a surfactant solution. In these experiments, the MD system was operated with the feed stream consisting of a base 3.5 wt % NaCl solution. The system was run for 2 h. Next, SDS was added such that specified concentrations of 0.1, 0.2, and 0.4 mM were obtained in the feed solution. The system was operated at each SDS concentration for 1 h. The permeate and conductivity were monitored every 10 min.
Finally, the system was tested with a 3.5 wt% NaCl solution containing 1000 ppm crude oil and 100 ppm SDS. The concentration of SDS was lower than the critical micelle concentration (8.2 mM) [26 (link)]. Flux and conductivity were recorded every 10 min.

The tissue myeloperoxidase activity assay was performed as an index of neutrophil recruitment, as previously described [26 (link)]. The test was repeated twice, with at least 5 mice from each group and each time point included from each experiment. Briefly, the skin was cut and weighed prior to homogenization in a 0.5% hexadecyltrimethylammonium bromide phosphate-buffered (pH 6.0) solution using a polytron PT1300D homogenizer (Kinematica, Lucerne, Switzerland). The homogenates were centrifuged at 14,000 rpm for 5 min at 4 °C in a microcentrifuge and five aliquots of each supernatant were transferred into 96-well plates, followed by the addition of a 3, 3′-dimethoxybenzidine and 1% hydrogen peroxide solution. Standard dilutions of pure myeloperoxidase were also tested for their activity to construct a standard curve (OD as a function of units of enzyme activity). Optical density readings at 450 nm were taken at 1 min (which corresponds to the linear portion of the enzymatic reaction) using a Spectra Max Plus plate reader using SOFTmax Pro v. 3.0 software (Molecular Devices Corp., Sunnyvale, CA, USA). Myeloperoxidase activity was expressed as units of enzyme per gram of tissue.