Polycarbonate membrane filter

l-α-Phosphatidylcholine from soybean and detergent used in this study were purchased from Sigma-Aldrich (Taufkirchen, Germany). Lipid mixtures solubilized in chloroform were first transferred to a round-bottom flask. A thin lipid film was formed by rotary evaporation under a controlled vacuum. The flask was then placed in a vacuum chamber overnight. The lipids were rehydrated in 1 mL of assay buffer A (50 mM HEPES-KOH, pH 7.5, 50 mM NaCl) to a final concentration of 40 mg/mL. The mixture was vortexed for 15 min to form multilamellar vesicles. The multilamellar vesicle solution was passed at least 21 times through an Avanti Polar Lipids (Alabaster, AL, USA) mini extruder holding a 200-nm Whatman polycarbonate membrane filter (Florham Park, NJ, USA) sandwiched with two filter supports on each side. The resulting unilamellar liposome solution was used for expression or water permeability experiments.

Tachyzoites of the 76 K strain were collected from an infected HFF T25 flask and purified by sequential syringe passage with 17-gauge and 26-gauge needles and filtration through a 3 µm polycarbonate membrane filter (Whatman). Brain cells that were grown and matured in Neurobasal A medium for 14 days were infected by the parasite. For that, the correct amount of tachyzoites was resuspended in 50 µl of Neurobasal A medium and then added onto the brain cell culture. Approximately 2 × 10⁵ brain cells were present in a well of a 24-well plate. Each well was infected by 3 × 10⁴ tachyzoites to a multiplicity of infection of one parasite for around seven cells. The infected culture was maintained at 37°C in a humidified 5% CO₂ incubator for the duration of the experiment without adding media to avoid disturbing the brain cell culture. A typical experiment yielded 2.5 × 10⁴ cysts per well of a 24-well plate (around 12.5% of the 2 × 10⁵ brain cells).

Lungs were dissected from mouse embryos in cold sterile Hanks’ Balanced Salt Solution under a stereomicroscope. The lungs were randomized by size and obvious outliers were excluded. The lungs were placed on a polycarbonate membrane filter (Whatman) floating on 1 ml of serum-free BGJb medium (Invitrogen) with penicillin/streptomycin in 60 mm Center-well Organ Culture Dishes (BD Falcon). The lung explants were positioned at the gas–liquid interface on the membrane filters. The Cultures were kept at 37°C in 5% CO2 and media was changed daily.

Liposomes were prepared as previously described (10 (link)). Briefly, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, succinyl-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl]), mPEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphorylethanolamine), and GT3 were mixed in chloroform, evaporated under N₂ flow, and lyophilized overnight. Lipid film was hydrated in phosphate-buffered saline (PBS) at 65°C for 1 h, and the lipid dispersion was extruded through a 0.1-μm-pore Whatman polycarbonate membrane filter at 65°C. Liposome size distribution and ζ-potential at 25°C in PBS, pH 7.4, were determined by dynamic light scattering using Zetasizer Nano-ZS (Malvern). Further ⁶⁴Cu labeling was performed by adding ⁶⁴Cu-CuCl₂ to the liposome at pH 5.5 with 0.2 M sodium acetate buffer at 50°C. The reaction was monitored by instant thin-layer chromatography silica gel paper using a 5 mM diethylenetriaminepentaacetic acid solution.

In order to determine the efficacy of the tested CHL and PEI combinations on bacteria embedded in biofilm matrix, P. aeruginosa was grown as a colony biofilm as previously described [34 (link)] with some modifications. Briefly, 10 µL from a diluted overnight culture (see above) were transferred to a sterilized polycarbonate membrane filter (25 mm diameter, 0.8 µm pore size, Whatman, Shrewsbury, MA, USA) placed on BHI agar plate. Plates were incubated at 37 °C for 4 days. Filters with the biofilm were subsequently transferred to a 6-well plate: a well for the sterility control, the membrane without the biofilm, a well for the positive control, membrane with biofilm on BHI agar without any antimicrobials and the rest four wells containing BHI agar mixed with different concentrations of CHL (2000, 1000, 500 and 250 µg mL⁻¹) and fixed concentration of PEI (100 µg mL⁻¹), as depicted in Figure 3. The plate was covered with a transparent seal, the lid was added, and the covered plate was incubated for 1 h at 37 °C in darkness to allow the diffusion of CHL and PEI into the agar through the membrane. Afterward, the plate was exposed to the red light. For viable cell enumeration, the biofilm biomass was resuspended in 10 mL phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) by vigorous vortexing and serially diluted.