Minisart nml syringe filter

Planktonic P. aeruginosa cells grown at 37°C were concentrated to 10¹⁰ CFU mL^-1 (5 000 × g, 15 min, 20°C). Tenfold dilutions of the concentrated bacterial suspensions were prepared in 50 mM morpholinopropane sulfonic buffer (MOPS; Fisher scientific, Belgium) containing F-CARV and E-CARV (at the MICs). K⁺ concentrations were measured at the time 0, 5 and 10 min in a tenfold dilution of the concentrated bacterial suspension filtrate (0.2 μm, Sartorius™ Minisart™ NML Syringe Filters, France) before contact with the antimicrobial solutions. After the exposure of the bacterial suspension cells to the F-CARV and E-CARV solutions, samples (4 mL) were filter sterilized at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 30 and 40 min. MOPS buffer with DMSO was used as control for F-CARV and without DMSO for E-CARV. The concentration of K⁺ in the filtrate samples was measured using a Varian SpectrAA 55/B atomic absorption spectrometer in flame emission mode (slit 0.7 nm high; wavelength 766.5 nm; air-acetylene flame).

The human plasma samples were filtered (Minisart NML syringe filters, pore size: 0.8 μm; Sartorius) and diluted with 1X phosphate‐buffered saline without calcium and magnesium buffer (Gibco) at 1:3 (v/v) ratio and processed for EV isolation by a charge—based isolation method as described before (Notarangelo et al., 2020 (link), 2019 (link)). The diluted plasma was recovered and processed for cfDNA isolation by QIAmp Circulating Nucleic Acid kit (Qiagen) (Lee et al., 2018 (link)).

In vitro release studies through a dialysis membrane (regenerated cellulose, Spectra/Por molecular porous membrane tubing, 12–14 KDa; Spectrum Laboratories, Inc., Piscataway, NJ, USA) were performed with Gummer-type diffusion cells.
Briefly, 200 mg of ETZ-SPMs was added in the donor compartment in direct contact with the membrane. The receptor compartment consisted of 5.0 mL of pH 7.4 (phosphate buffered saline, PBS) added to 0.01% Brij 98 maintained at 37 °C and stirred at 600 rpm. Brij 98 was added to improve the ETZ solubility in the receiving fluid [29 (link)].
At predetermined time intervals, 5 mL of the samples was withdrawn and replaced with the same amount of fresh receiving fluid. All the experiments lasted 24 h and were performed in triplicate. The amount of ETZ in the samples was determined by HPLC after filtration through cellulose acetate filters (0.22 um pore size, Minisart^® NML Syringe filters, Sartorius, Florence, Italy).

pLx302-SOX2 was generated using Gateway system (Invitrogen) according to the manufacturer's instructions. SOX2 was cloned into pDONOR221 (Invitrogen) from pMXs-Sox2 (Addgene) plasmid by BP Gateway clonase (Invitrogen) to generate entry clones. Using LR Gateway clonase (Invitrogen) SOX2 was cloned into the destination vector pLx302 (a gift from Dr. David Root (Yang et al., 2011 (link)); Addgene plasmid 25896). Packaging cells (HEK-293T) were co-transfected with the packaging vector p8.91, the envelope plasmid pMDG (gifts from Dr. Didier Trono, University of Geneva, Switzerland) and pLX302-SOX2 as previously described (Besnier et al., 2002 (link)). Following 72 h the HEK293-T medium containing the virus was collected, filtered through a 0.45 μm Minisart NML Syringe Filter (Sartorius) and stored at − 80 °C for later transduction.

All patients received ferumoxtran-10 intravenously in a weight-adapted dose of 2.6 mg/kg of body weight 24–36 h before the MRI scan. Ferumoxtran-10 was diluted in 100 mL of 0.9% NaCl solution and administered via drip infusion using a 0.22-μm-pore filter (Minisart NML syringe filter, catalog no. 16534-k; Sartorius AG). The infusion was performed at a slow rate of 1 mL/min at the start, increasing to 4 mL/min. The infusion duration was approximately 45 min and supervised by radiologists. MRI was performed using a 3-T MRI scanner (Magnetom Skyra or Trio; Siemens Healthineers). The imaging area included the pelvis from the pubic bone to the aortic bifurcation. The MRI protocol consisted of an isotropic 3-dimensional T1-weighted gradient-echo sequence (repetition time, 6.5 ms; echo time, 2.5 ms; flip angle, 10°; and spatial resolution, 0.9-mm isotropic) and an isotropic 3-dimensional iron-sensitive T2*-weighted gradient-echo sequence with fat saturation (multiple-echo data image combination, with repetition time, 21 ms; echo time, 12 ms; 3 combined echoes; flip angle, 10°; and spatial resolution, 0.85-mm isotropic).