Fluostar omega microplate reader

The antibacterial capacity of AOS and COS against C. difficile was determined by analyzing the MIC as previously described [41 (link),78 (link)]. NDOs were serially diluted in 96-well U-bottom polypropylene plates (Corning Costar, Cambridge, MA, USA) until reaching the final volume of 100 μL. Subsequently, 100 μL of bacterial inoculums (C. difficile) with OD₆₀₀ = 0.2 (approximately 4.10⁷ colony-forming units [CFU]/mL) were added to the serially diluted NDOs [79 (link),80 (link)]. Starting OD was measured at 600 nm with a FLUOstar Omega microplate reader (BMG Labtech GmbH, Ortenberg, Germany). The plates were incubated anaerobically in a vinyl anaerobic chamber (Coy labs) overnight at 37 °C. Then, 100 μL of culture medium was transferred to 96-well F-bottom polystyrene microtiter plates (Corning Costar, Cambridge, MA, USA), and the signal was measured at 600 nm with a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). Bacteria growth in BHIs without treatment served as a positive control, and BHIs containing different concentrations of COS or AOS were used as a negative control. The starting OD was subtracted from the final OD. Finally, the MIC was considered as the lowest concentration that inhibits bacterial growth in comparison to the positive control groups [42 (link)].

HEK293 cell were plated at a density of 4 × 10⁴ cells /96 well and simultaneously transiently transfected with the following vectors: pcDNA3.1-TRPV1-YFP (0.5 μg/30 wells) and/or with pcDNA3.1-ARMS (2.5 µg/30 wells) and/or pRFP-C-RS-sh (1.75 µg/30 wells) using X-treme GENE HP DNA Transfection Reagent (Roche, Mannheim, Germany) in a 3:1 ratio. At 48 h post transfection, HEK 293 cells were washed and loaded with the fluorescent calcium indicator dye Fluo 4 AM (1 μM, Invitrogen, Darmstadt, Germany) in DMEM without phenol red for 60 min. Baseline calcium flux was measured with FLUOstar^® Omega Microplate Reader (BMG LABTECH, Ortenberg, Germany): bottom reading, 7 × 7 well scanning, 485 BP/510, orbital shaking and at 37 °C. TRPV1 was activated by capsaicin (0.1 nM–10000 nM) and calcium flux was measured again with FLUOstar^® Omega Microplate Reader (BMG LABTECH, Ortenberg, Germany).

B-RAF-V600E enzyme assay was performed using a luminescence-based kinase assay kit (BPS Bioscience) according to the manufacturer’s instructions. Briefly, a master mix with a final volume of 25 µL containing kinase buffer, 500 µM ATP, RAF substrate, and water was added to each well of a 96-well white bottom plate. DMSO (5 μL) or various concentrations of CB-RAF600E-1 (0.1 nM to 10,000 nM in log scales) were added to the wells, and 2 ng/µL B-RAF-V600E was added to initiate the reaction. Suitable blanks contained 5 µL of kinase buffer without the enzyme. The plate was incubated at 30 °C for 45 min in the dark. Kinase-Glo Max reagent (50 µL) was added to each well and incubated for 15 min at 25 C^o. Luminescence was measured using a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). The Akt inhibition assay was performed with 0.1 nM to 10,000 nM in log scale concentrations of compound using the Z'-LYTE™ kit (Invitrogen) according to the manufacturer’s instructions. The ratio of fluorescence emission at 520 nm to coumarin emission at 445 nm was used to quantify the reaction progress. Fluorescence energy transfer (FRET) was measured using a FLUOstar Omega microplate reader (BMG Labtech). GraphPad Prism was used to calculate IC₅₀ values for both enzyme assays.

EGI-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and TFK¬1 were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum and 1% Penicillin-streptomycin (100 IU/mL and 100 g/mL, respectively). All media and supplements were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cells were incubated at 37 °C with 5% CO2. Cells were incubated with 20 µg/ml Irinotecan or left untreated.
For the cell viability and cytotoxicity assays, 5,000 TFK-1 or EGI-1 cells per well were seeded into a 96-well plate in quadruplicates. Cells were incubated with 20 μg/ml Irinotecan or left untreated for up to 3 days and measurements were performed at 0, 48 and 72 h. For cytotoxicity measurement, CellTox™ Green Cytotoxicity Assay (Promega, Madison, WI, USA) was used. CellTox™ Green was 1:1000-diluted with assay buffer, added to the cells and incubated for 15 min at RT in the dark and measured (485 nm Ex/520 nm Em) with an Omega FLUOstar Microplate Reader (BMG LABTECH, Ortenberg, Germany). For cell viability measurement, growth medium containing 10% Resazurin (R&D Systems, Minneapolis, MN, USA) was added to the cells, incubated for 1 h at 37 °C and measured (544 nm Ex/590 nm Em) with an Omega FLUOstar Microplate Reader (BMG LABTECH). All experiments were repeated three times in quadruplicates for both cell lines.

The ability of SP-A (or rfhSP-A), PMB, PMBN, and combinations thereof to permeabilize the outer and cytoplasmic bacterial membranes was studied in live bacteria by quantifying the internalization of the impermeant fluorescent Sytox Green, since its fluorescence increases when binding to bacterial DNA (31 (link)). For the measurement of Sytox Green influx, the probe (1 μM) was added to 1 ml of bacterial suspension (2x10⁷ CFU/ml) in LTM and the sample was incubated for 15 min in darkness at room temperature. Then, the fluorescence of the Sytox Green/bacterial suspension mixture was monitored for 4 hours in a FLUOstar Omega microplate reader (BMG LabTechnologies, Ortenberg, Germany) at excitation and emission wavelengths of 485 and 520 nm, respectively. PBS was used as a negative control, whereas ethanol (70%) was used as a positive control. Background fluorescence was measured in non-labeled bacteria.

The amount of global, genome-wide DNA methylation was quantified using the Methylamp global DNA methylation quantification kit (Epigentek) according to the manufacturer's instructions. In this assay, 5-methylcytosine-modified genomic DNA is recognized by 5-methylcytosine antibody, and the bound DNA is quantified in a fluorometric assay. Positive (methylated) and negative (unmethylated) control DNA was supplied with the kit. Fluorescence was measured on the FLUOstar Omega microplate reader (BMG Lab Technologies). The amount of DNA methylation (percent methylation) was calculated using the following formula: percent methylation = [OD (sample − negative control) × GC content]/[OD (positive control − negative control) × 10] × 100%.

The ability of LL-37, SP-A, rfhSP-A, and combinations thereof to permeabilize the outer and cytoplasmic bacterial membranes was studied in live bacteria by quantifying the internalization of the impermeant fluorescent Sytox Green, since its fluorescence increases when binding to bacterial DNA. For the measurement of Sytox Green influx, the probe (1 μM) was added to 1 ml of bacterial suspension (2x10⁷ CFU/ml) in PBS and the sample was incubated for 15 min in darkness at room temperature as described in (25 (link)). Then, the fluorescence of the Sytox Green/bacterial suspension mixture was monitored for 4 hours in a FLUOstar Omega microplate reader (BMG LabTechnologies, Ortenberg, Germany) at excitation and emission wavelengths of 485 and 520 nm, respectively. PBS was used as a negative control, whereas ethanol (70%) was used as a positive control. Background fluorescence was measured in non-labeled bacteria.