Infinite m1000 pro

Population survival in the presence of phage CR30 were measured in 750 μL cultures in PYE at 30°C with shaking (244.5 rpm, 2.5 mm radius) in an Infinite Pro M1000 microplate reader (Tecan, Männedorf, Switzerland). Fresh overnight cultures were diluted to an OD₆₆₀ of 0.03 (Ultrospec 10 Cell Density Meter, GE Healthcare Life Sciences, Pittsburgh, PA) in fresh PYE, distributed into a 48-well flat bottom plate (Genesee Scientific, San Diego, CA), and outgrown for 2 hours in an Infinite Pro M1000 microplate reader (Tecan, Männedorf, Switzerland). Phage lysate was added to a final concentration of 2.6x10⁶ pfu/mL with a predicted multiplicity of infection (MOI) of 0.005–0.01 and the plate was returned to the reader. Throughout the duration of the experiment, optical density measurements were taken every 15 min at an OD₆₆₀ for 15 hours.

About 200 mg of powdered cookies were extracted with 80% methanol according to methodology described by Przygodzka et al. [26 (link)]. Then extracted samples were stored in −80 °C before prior analysis of antioxidant and reducing capacity (DPPH, PCL, and FRAP assays).
Therefore, DPPH assay was established to measure antioxidants (extracted from cookies) scavenging ability against DPPH radicals [39 (link)]. The measurement were performed at a microplate reader (Tecan M1000 Infinite PRO) with the wavelength established at 517 nm. Results were presented as mmol Trolox per gram of sample. PCL method was used to measure ability of antioxidants from cookies to scavenge superoxide anion radicals (O₂^−•). The measurement was performed using PHOTOCHEM apparatus (Analytik Jena, Jena, Germany) according to protocols elaborated by Zieliński et al. [40 (link)]. The extracts of cookies were determined in two methodologies: for lipophilic antioxidants (PCL ACL) and hydrophilic (PCL ACW) methodology extracts of were expressed as µmol Trolox per gram of sample. The reducing power was determined using FRAP assay according to Horszwald and Andlauer [39 (link)]. The mixture’s absorbance was measured at 593 nm after 5 min reaction with microplate reader (M1000 Infinite PRO, Tecan, Männedorf, Switzerland). The FRAP method is based on the reduction of ferric ion by antioxidant compounds.

According to a slightly modified procedure already reported in the literature [33 (link)], the generation of ROS was determined using an oxidation-sensitive fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, D6665; Sigma-Aldrich, St. Louis, MO, USA). Briefly, viable cells were seeded in a black 96-well cell culture plate (Costar, Sigma-Aldrich, St. Louis, MO, USA) and after 24 h were incubated with different concentrations (0.1–100 µM) of each compound for 1 h at 37 °C in 5% CO₂. DCFH-DA in medium without serum was added directly to each well at a final concentration of 25 μM, and the plate was incubated at 37 °C for 30 min at 37 °C in 5% CO₂. After washing using PBS, 100 μM H₂O₂ in medium without serum was added to each well and the cells were incubated for an additional 30 min. The formation of fluorescent dichlorofluorescein (DCF) due to oxidation of DCFH in the presence of ROS, was read at 530 nm using a microplate reader Tecan Infinite M1000 Pro (Tecan, Cernusco S.N., Italy) and DMSO medium was used for control cells. The results are expressed as mean ± SEM of at least three independent measurements in triplicate.

100 mg of liver tissue were lysed in 2.5 ml of lysis buffer A (10 mM KCl, 10 mM Tris–HCl pH 8.0, 2 mM MgCl₂ and 0.05% NP-40) with a protease inhibitor cocktail (Roche #04693116001). Following incubation on ice for 20 min, tissues were lysed using a tight-fitting douncer with 40 strokes, followed by centrifugation at 13 000 rpm spin at 4°C for 10 min to remove cell debris. A sucrose gradient was created by adding 2 ml of 36, 29, 22 and 15% sucrose to Beckman ultracentrifuge tubes and then 2 ml of the supernatant sample were added to the sucrose gradient to make up the final volume to 10 ml. A discontinuous gradient of 15–36% was formed and the tubes were centrifuged at 40 000 rpm for 5 h at 4°C (Beckman SW 41 Ti swing-bucket rotor). From the top of each gradient, 800 μl gradient fractions were collected to yield a total of 12 fractions. The Alexa fluorescence intensity of the fractions was measured using TECAN infinite M1000 Pro (TECAN).

To assess the activity of our compounds as FAAH inhibitors, 96-well black flat-bottom microtiter NBS plates (COSTAR flat black) were used. The assay was conducted in a total volume of 200 µL, with different concentrations of each tested compound (in triplicate) being preincubated for 10 min at room temperature in an appropriate fluorometric assay buffer (tris-HCl 125 mM, Na₂EDTA·2H₂O 1 mM, pH = 9.0) also containing the enzyme (FAAH Human recombinant, Cayman Chemical, Ann Arbor, MI, USA), while the plate was being kept in orbital shaking. Following this, the substrate (7-amino-4-methyl-2H-1-benzopyran-2-one-5Z,8Z,11Z,14Z-eicosatetraen-amide, AMC-AA, 5 µM final concentration) was added, and the plate was incubated for 2 h at 37 °C in a TECAN infinite M1000Pro plate reader (Tecan, Männedorf, Switzerland), reading the signal from each well every 30 s (λex = 340 nm, λem = 450 nm) and thus expressing FAAH activity as relative fluorescence units (RFU). The percent inhibition for each tested compound was calculated using control wells lacking the inhibitor and blank wells lacking both inhibitor and enzyme. IC₅₀ values were calculated using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA) and are expressed as mean ± SEM of at least two independent measurements performed in triplicate.

Three hundred thousand (300,000) Huh7 cells were cultured in 6-well plates and transfected the following day at 70–80% confluency using polyethylenimine [97 (link)] (PEI; Sigma, ON, Canada, cat. no. 919012) or lipofectamine 2000 [98 (link)] (Invitrogen, ON, Canada, cat. no. 11668019), as described previously, with slight modifications [33 (link)]. The EBOV miRNA mimics and/or plasmids to be transfected, as well as the transfection reagents, were diluted in Opti-MEM^® (Invitrogen, Burlington, ON, Canada, cat. no. 31985062). Forty-eight (48) h after transfection, cells were washed with PBS and lysed with 500 μL of the passive lysis buffer. Luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA, cat. no. E1980) on a luminometer (TECAN INFINITE M1000 PRO, Tecan Austria GmbH, Grödig, Austria), according to the manufacturer’s instructions. Rluc activity was expressed relative to expression of the internal control Firefly luciferase (Fluc). Rluc expression was further normalized to the control in which cells were co-transfected with synthetic unrelated miRNA mimic, elsewhere referred to as mock control. All assays were conducted in triplicate in a 96-well format.