Synergy h1 hybrid

The levels of mitochondrial reactive oxygen species (ROS) generation were measured by using MitoSOX™ Red mitochondrial superoxide kit (M36008, Thermo Fisher). Experiments were conducted according to the manufacturer’s instructions. Briefly, S2 cells were harvested, washed twice with PBS, resuspended in 5 μM MitoSOX reagent working solution and then incubated at 25 °C for 20 min in darkness. The fluorescence intensities (excitation/emission [Ex/Em] = 510/580 nm) were measured on a multi-mode microplate reader (Synergy H1 Hybrid, BioTek).

The synthesized peptides were screened for antifungal activity against the molds A. pullulans (Ap) and Rhodotorula sp. (Rh) (both obtained from Professor Arne Tronsmo, The Norwegian University of Life Sciences, Ås, Norway) and the yeast C. albicans (Ca, ATCC 10231) as previously described [13 (link)]. In short, fungal spores were grown in potato dextrose broth media (Difco) containing 2% D(+)-glucose (Merck, Darmstadt, Germany) at 25–30 °C while shaking at 200 rpm overnight. The cultures were diluted with a dextrose media containing glucose to a concentration of approx. 4 × 10⁵ spores/mL. Aliquots of the cultures (50 µL) were transferred to 96 well microtiter plates preloaded with the synthetic peptides (50 µL) in two-fold serial dilutions (256 to 1 µg/mL). Polymyxin B and CHX (both with concentrations ranging from 12.5 to 0.09 μg/mL) served as positive antibiotic controls. The microtiter plates containing the fungal spores and the test peptides were incubated at room temperature for 48 h and OD₆₀₀ was recorded using a Synergy H1 Hybrid microplate reader system (BioTek, Winooski, VT, USA). MIC was defined as the lowest peptide concentration showing an optical density less than 10% of the negative (growth) control. All experiments were performed in triplicate.

Hyclone^TM trypan blue stain and BCA protein assay were purchased from ThermoFisher Scientific and used as purchase. A ThermoScientific microcentrifuge was used in the process of CG production from U937 monocytes. Cyanine 3 (Cy3) NHS monoester dyes were purchased from Kerafast and used as per manufacturer’s recommendations. Cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids. A BioTek Synergy H¹ Hybrid microplate reader was used for fluorescence measurements. Extrusion was performed with a Genizer Jacketed Gextruder (10 mL).
DOPC and cholesterol used in the production of nCVTs and liposomes were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Filter membranes used during extrusion were purchased from GE Healthcare Life Sciences (Marlborough, MA, USA). DNA isolation was performed by using the DNeasy Blood & Tissue Kits from Qiagen (Hilden, Germany), following the manufacturer’s protocol. Measure-IT^TM High-Sensitivity Nitrite Assay Kit was purchased from ThermoFisher Scientific (Waltham, MA, USA). ELISA kits for measuring interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) were obtained from Biolegend (ELISAmax™, San Diego, CA, USA).

hGDF cells were seeded on top of each specimen at a density of 1 × 10⁴ cells/well. The hGDFs were incubated in direct contact with the disc for 1, 3, and 5 days. At the end of each incubation period, a solution of 0.5 mg/mL MTT (Sigma Aldrich, St. Louis, MO, USA) was added to each well and then the cells were incubated for 4 h at 37 °C and 5% CO₂. A solubilization solution was added into each well to dissolve the insoluble formazan. The spectrophotometrical absorbance of the samples was measured at 650 nm using a microplate reader (Synergy H1 Hybrid BioTek Instruments, Winooski, VT, USA). Five replicates and three independent analyses were assessed for MTT assay.