Bmg fluostar omega

SNCA Promoter fragments and assay conditions were as previously described [31 (link)]. SH-SY5Y cells were performed in 24-well plates seeded at 5 × 10⁴ cells/well 24 h prior to transfection. Transfections of promoter constructs in pGL Basic (with firefly luciferase activity) were performed using FuGENE HD transfection reagent (Promega), as per manufacturer’s instructions. To control for variation in transfection efficiency among replicates, promoter constructs were co-transfected with the Renilla luciferase vector, pRL-TK (Promega). At 24 h post transfection, SH-SY5Y cells were harvested and firefly and Renilla luciferase chemiluminescence were measured while using the Dual-Luciferase Reporter Assay System (Promega) in a BMG FLUOstar Omega plate reader (BMG Labtech GmbH, Offenburg, Germany). Luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity.

The cytotoxicity profile of the formulation without oil (ENE), nanoemulsion containing copaiba oil (CNE), and free copaiba oil (FCO) were measured by the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] method (Sigma-Aldrich, Missouri, USA). This experiment was performed in three independent events.
Twenty-four hours before treatment, 5x10³ VERO E6 cells were plated in a 96-multi-well plate. On the following day, CNE and FCO were added at concentrations ranging from 5.6, 11.2, 22.5, 45, 90, 180, and 360 μg/mL. Since the tested concentrations were based on copaiba oil, the ENE treatment was performed with the same volume used for each concentration of CNE. Cells were incubated for 24, 48, and 96 hours for VERO E6, in triplicate. After these times, the culture medium was removed from each well and exchanged for MTT solution (1 mg / mL in 100 μL of culture medium without supplementation) (Sigma-Aldrich, Missouri, USA). Cells were incubated for 30 minutes at 37°C. After the formation of the formazan crystals, the MTT solution was removed and 100 μL of DMSO (Synth, Sao Paulo, Brazil) were added per well. The absorbance was then measured at 572 nm, using a plate spectrophotometer (FLUOstar Omega/BMG LABTECH, Offenburg, BW, DE). The maximum non-cytotoxic concentration was determined considering a cut-off of 80% of cell viability.

For Western blot analysis, protein concentrations were measured by Quick Start^TM Bradford Protein assay (Bio-Rad) using the plate reader Fluostar Omega BMG (BMG Labtech) and the corresponding MARS Data Analysis Software (BMG Labtech). Proteins were separated on NuPAGE®Novex® 4–12 % Bis-Tris Protein Gels (Life Technologies) followed by transfer onto a PVDF membrane (GE Healthcare) in a wet blotting chamber. Western blot analysis was performed using mouse anti-LDL receptor (1:500; NovusBiologicals); mouse anti-Alix (1:1000; BD Bioscience); rat anti-HA 3F10 (1:1000; Roche); mouse anti-GAPDH 6C5 (1:5000; Abcam); mouse anti-Hsc/Hsp70 N27F3-4 (1:1000; ENZO); mouse anti-VSV-G 5D4 (1:1000; Sigma); rat anti Sup35 M domain (1:10; hybridoma supernatant);^{88 (link)} rabbit anti-Tau ab64193 (1:1000; Abcam); rabbit anti-Flotillin 1 ab133497 (1:1000; Abcam); mouse anti-SARS-CoV-2-spike S GTX632604 (1:1000; GeneTex); rabbit anti-hACE2 ab15348 (1:1000; Abcam); rabbit anti-clathrin heavy chain (1:1000; Abcam); rabbit anti-GFP (1:5000; Abcam). The membrane was incubated with Pierce^TM ECL Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer´s recommendations and imaged with the Imaging system Fusion FX (Vilber Lourmat).

The toxicity of the (p-BthTX-I)₂K peptide in BHK-21 and Vero cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described [28 (link)] with modifications. The cells were seeded in 96-well culture plates (TPP, Trasadingen, Switzerland) (5 × 10³ cells/well) for 24 h. Then, the cells were incubated with the (p-BthTX-I)₂K peptide at concentrations of 1.6, 3.1, 6.3, 12.5, 25, 50, and 100 µM. After 24 h (BHK-21) or 48 h (Vero), the medium containing the peptide was aspirated, and 100 µL of MTT (Sigma–Aldrich, St. Louis, MO, USA) diluted in DMEM (Cultilab, Campinas, SP, Brazil) (final concentration: 1 mg/mL) was added to the cells. After 30 min of incubation at 37 °C, the medium containing MTT (Sigma–Aldrich, St. Louis, MO, USA) was removed, and 100 µL of dimethylsulfoxide (DMSO) (Synth, Diadema, SP, Brazil) was added to each well of the plate, which was rotated at 200 rpm for 5 min. The absorbance was measured at a wavelength of 572 nm with a plate reader (FLUOstar Omega/BMG LABTECH, Ortenberg, Germany).