1000 cells/well were incubated in HMI-9 medium for 48 h with an optimized range of palmostatin B or 2-BP concentrations in triplicate. The cell viability dye Alamar Blue was added at 44 h and incubated for 4 h so a change in fluorescence could be measured at 520 nm emission and 545 nm excitation on a FLUOstar Omega spectrophotometer (BMG Labtech GmbH, Ortenberg, Germany) [34 (link)]. All data points were normalized to the HMI-9 medium control wells for relative fluorescence. A non-linear variable slope four parameter curve fit was used to calculate IC50 values.
Fluostar omega spectrophotometer
Manufactured by BMG Labtech
Sourced in Germany, United States
The FLUOstar Omega is a high-performance spectrophotometer designed and manufactured by BMG LABTECH. It is capable of measuring absorbance, fluorescence, and luminescence in a wide range of applications. The instrument features a monochromator-based optical system, allowing for precise wavelength selection and accurate measurements.
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Lab products found in correlation
Celltiter aqueous one solution cell proliferation assay, by Promega (3 mentions)
Innova 40r shaking incubator, by Eppendorf (2 mentions)
96 well tissue culture plate, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Glycerol quantification kit, by Merck Group (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Optima 2000 dv, by PerkinElmer (1 mentions)
Crystal violet solution, by Merck Group (1 mentions)
Yeast extract, by BD (1 mentions)
Anti human igg hrp, by Merck Group (1 mentions)
O phenylenediamine dihydrochloride, by Merck Group (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Cut off filters, by Merck Group (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Np klh, by Biosearch Technologies (1 mentions)
Hrp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
49 protocols using fluostar omega spectrophotometer
1
Cytotoxicity Assay of Palmitoylation Inhibitors
2
Quantifying Cell Viability via MTS Assay
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Cell viability was assessed via a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, which utilizes the ability of mitochondria to reduce a substrate (MTS) into a soluble formazan product. For the assay, the CellTiter 96 AQ ueous One Solution cell proliferation colorimetric assay kit was used, in accordance with the manufacturer's instructions (Promega, Madison, WI, USA). The MTS assay was performed using 96-well plates containing the treated cells. Following the treatment paradigms, 20 µl MTS reagent was added to each well, and the plates incubated at 37 °C for 3 h. The absorbance of the solution was read at 490 nm using a FLUOstar Omega spectrophotometer (BMG LabTech, Germany). Experiments were performed in triplicate. Results were expressed as a percentage of control. The MTS assayderived data were validated by means of traditional trypan blue dye exclusion. For this, an aliquot of cell suspension in PBS was mixed with an equal volume of trypan blue solution (0.4% in PBS). In total, 10 µl of cells were added to a Neubauer haemocytometer chamber and the average number of cells was counted by an observer blind to treatment protocol. Cell death was expressed as percentage trypan blue positive cells per total population of stained and unstained cells.
3
Quantification of FFA and Glycerol in Skeletal Muscle
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For FFA and free glycerol determination, 40 mg of Skm tissue were homogenized in 1 ml of 2-isopropanol in a TissueLyser (Qiagen). Five microliters of Skm extracts were used for TAGs quantification using the Glycerol Quantification Kit (Sigma-Aldrich) and a FLUOstar-Omega spectrophotometer (BMG Labtech). All results were adjusted for exact protein content.
4
Geochemical Analyses of Methane, DIC, and Ions
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Methane was measured using a Gas Chromatograph (GC) (310C, SRI Instruments, United States) with a flame ionization detector. The carbon isotope composition of methane was determined using a coupled pre-concentration GC/Isotope Ratio Mass Spectrometer system (GC/IRMS) (Thermo Fisher Scientific, Germany) (Rice et al., 2001 (link)). The δ13C values were reported vs. the Vienna Pee Dee Belemnite standard (VPDB). DIC was measured on the same GC/IRMS system without pre-concentration (Torres et al., 2005 (link)). Sulfate was measured by an IC-2500 ion chromatography system (Dionex Corporation, United States), NH4+ was determined by a FLUOstar Omega spectrophotometer (BMG LABTECH, Germany) based on the salicylate-hypochlorite method (Bower and Holm-Hansen, 1980 (link)), and calcium was measured by inductively coupled plasma optical emission spectrometry (ICP-OES) (Perkin Elmer Optima 2000DV, United States).
5
Antioxidant Capacity Determination Methods
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Antioxidant capacity was determined using four different methods; TEAC (2,2′-azinobis-3-ethyl-benzo-thiazoline-6-sulfonic acid) using the methodology of Re et al. [22 (link)]; ORAC (oxygen radical absorption capacity) according to the methodology of Robles-Sánchez et al. [23 (link)]; DPPH radical scavenging activity according to Brand-Williams et al. [24 (link)]; and the FRAP assay according to the methodology of Benzie and Strain [25 (link)]. Results of all these methods were expressed as mg of Trolox equivalents (TE)/g dw. Antioxidant capacity analyses were performed in triplicate; absorbances were read in a FLUOstar Omega spectrophotometer (BMG Labtech).
6
Cytotoxicity and Clonogenic Assay
7
Growth Assay of Acinetobacter baumannii
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Acinetobacter baumannii AB5075_UW mutant derivatives were purchased from the Manoil Laboratory (30) and T26 transposon insertions confirmed by PCR. The A. baumannii ATCC 17978 czcA and czcE mutants were generated in our laboratory for a previous study (22) (See Tables S1 andS2 for list of strains and oligonucleotides used in the study, respectively). All chemicals were purchased from Sigma Aldrich (Australia) unless otherwise indicated.
A. baumannii strains were routinely grown in Luria Bertani broth (LB), containing 1% tryptone (BD Bacto), 0.5% yeast extract (BD Bacto) and 0.5% sodium chloride. For overnight culturing, a single colony from LB agar (1.5%) was used to inoculate 4 mL of LB medium. Overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.01 in 200 μL LB for growth assays or 20 mL LB for all other analyses. For growth assays, cultures in LB media were incubated at 37°C with shaking in a FLUOstar Omega Spectrophotometer (BMG Labtech), with the OD600 values presented. The 20 mL cultures used for all other analyses were incubated at 37°C in an Innova 40R shaking incubator (Eppendorf) at 230 rpm until they reached an OD600 of 0.7.
A. baumannii strains were routinely grown in Luria Bertani broth (LB), containing 1% tryptone (BD Bacto), 0.5% yeast extract (BD Bacto) and 0.5% sodium chloride. For overnight culturing, a single colony from LB agar (1.5%) was used to inoculate 4 mL of LB medium. Overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.01 in 200 μL LB for growth assays or 20 mL LB for all other analyses. For growth assays, cultures in LB media were incubated at 37°C with shaking in a FLUOstar Omega Spectrophotometer (BMG Labtech), with the OD600 values presented. The 20 mL cultures used for all other analyses were incubated at 37°C in an Innova 40R shaking incubator (Eppendorf) at 230 rpm until they reached an OD600 of 0.7.
8
Quantifying Flavonoid Content in Extracts
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The flavonoid content was determined based on the method described by Chen at al. [14 (link)], with slight modifications. The extracts were dissolved in absolute methanol. In a 2 mL Eppendorf tube, 100 μL of a sample was mixed with 430 μL of 5% NaNO2, followed by incubation for 5 min. After incubation, 30 μL of AlCl3 (10%) and 440 μL of NaOH (1 mol/L) were added to the reaction mixture, and the absorbance was read at 496 nm with a multimode microplate reader Fluostar Omega spectrophotometer (BMG Labtech, Chicago, IL, USA), using quercetin as the standard. The results were expressed as mg of quercetin equivalents (QE) per g of extract (mg QE/ge).
9
Quantifying Phenolic and Flavonoid Compounds
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Total phenolic compounds were quantified using the Folin–Ciocalteu method [20 (link)]; a standard curve of gallic acid was used to calculate these values, according to their absorbance at 765 nm. Data were expressed as mg gallic acid equivalents (GAE)/g dry weight (dw). Flavonoid content was determined using a standard quercetin curve; absorbance was measured at 496 nm and was expressed as mg of equivalents of quercetin (QE)/g dw [21 (link)]. Both analyses were performed in triplicate; absorbances were read in a FLUOstar Omega spectrophotometer (BMG Labtech, Durham, NC, USA).
10
Apigenin-mediated Oxidative Stress Assay
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RWPE-1 cells were plated at 1×105 cells per well in 96-well plates in appropriate culture medium. As cells reached to 75–80% confluence subsequently treated with different concentration of apigenin for 16 h and 200 µM H2O2 for 6 h. The treatment medium was removed and cells were washed with phosphate-buffered saline (PBS) and than exposed to PBS containing 10 µM 2′, 7′-dichlorofluorescein diacetate (DCF-DA), a dye that fluoresces when ROS are generated. The cells were incubated with DCF-DA for 20 min, after which fluorescence intensity was determined using FluoStar Omega Spectrophotometer (BMG Labtech) at 480 nm excitation and 560 nm emission as previously described [27] (link). The values, expressed in percentage arbitrary fluorescence units, were compared across treatment groups.
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