Wallac victor3 1420 multilabel counter

Liquid nitrogen frozen mCherry MAP Gc86 was thawed at 37 °C and was used to inoculate 5 ml of 7H9 broth (Difco laboratories, Franklin Lakes, NJ, USA) supplemented with 10 % oleic acid-albumin-dextrose-catalase (OADC; Sigma-Aldrich, St. Louis, MO, USA), 0.25 % v/v Tyloxopol (Sigma-Aldrich), 50 μg/ml kanamycin (Allied Laboratories, Wichita, KS, USA) and 2 μg/ml of mycobactin J (Allied Laboratories). The cultures were incubated at 37 °C, and once they reached a fluorescent intensity (FI) of 45000, equivalent to OD₆₀₀ = 0.8 [25 ], 5 ml aliquots were sub-cultured into 100 ml of media in a 250 ml sterile culture flask and incubated at 37 °C. When the cultures reached the logarithmic stage of growth (FI = 40000–50000 equivalent to OD₆₀₀ = 0.6–0.9), cells were centrifuged at 2000 × g for 30 min. The cells were re-suspended to reach FI = 60000 equivalent to OD₆₀₀ = 1.0 using Fig. 1, then to establish Colony Forming Units (CFU/ml) using Fig. 2 [25 ]. Quantification of fluorescence was based on the specific emission (587 nm) and excitation wavelengths (610 nm) for mCherry using the Wallac-1420 VICTOR3 Multilabel Counter (Perkin Elmer, Woodbridge, ON, Canada).Fig. 1

OD₆₀₀ versus fluorescent intensity of mCherry MAP Gc86 [25 ]

Fig. 2

OD₆₀₀ versus CFU of mCherry MAP Gc86 [25 ]

thiobarbituric acid reactive substances (TBARS), markers of lipid peroxidation, were evaluated using 100 µL of plasma, according to the method by [32 (link)]. Briefly, after the addition of 100 µL TCA, the resulting supernatant (160 µL) was added to 32 µL thiobarbituric acid, 0.12 M (Sigma-Aldrich, Milan, Italy city, country) and heated at 100 °C, for 15 min. The samples were then placed for 10 min in ice and centrifuged at 1600× g at 4 °C, for 10 min. The absorbance of the supernatants was measured at 532 nm by using a Wallac 1420 Victor3 Multilabel Counter (Perkin Elmer, Waltham, MA, USAmanufacturer, city, country). The amount of TBARS, expressed as µM, was calculated using a molar absorption coefficient of 1.56 × 10⁻⁵ M⁻¹ cm⁻¹.

TBARS were evaluated as an index of lipid peroxidation according the method by Dietrich-Muszalska et al. [16 (link)]. A total of 100 µL of serum was first deproteinized by adding 100 µL of TCA, then 160 µL was added to 32 µL of 0.12 M thiobarbituric acid (Sigma-Aldrich, Milan, Italy) in TRIS 0.26 M, and heated for 15 min at 100 °C. The reaction was stopped by placing the vials in an ice bath for 10 min and after centrifugation (at 1600× g at 4 °C for 10 min), the absorbance of the supernatant was measured at 532 nm (Perkin Elmer Wallac 1420 Victor3 Multilabel Counter).
TBARS content was calculated using a molar absorption coefficient of 1.56 × 10⁻⁵ M⁻¹ cm⁻¹ and expressed as µM.

Assays based on Benzie and Strain’s method were used to determine FRAP [28 (link)]. As previously described [25 (link)], serum samples (10 μL) were added to 90 μL of distilled water. A FRAP solution (300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl₃·6H₂O (10:1:1)) was freshly prepared incubated for 30 min at 37 °C. The ferric–tripyridyl–triazine complex is reduced into the ferrous form, developing an intense blue color. The absorbance value was measured at 595 nm using the Wallac 1420 Victor 3 Multilabel Counter (Perkin Elmer, Waltham, MA, United States). FeSO₄·7H₂O was used for the standard curve, FRAP was expressed as nmol/mg protein. Merck KGaA (Darmstadt, DA, Germany) supplied all reagents.

To assess the effect of PLX on cancer cells, a water-soluble tetrazolium salt (WST-1) based colorimetric assay was carried out as per manufacturer's protocol (Roche Applied Science, Indianapolis, IN, USA), to quantify cell viability as a function of cellular metabolism. Equal number of cells were seeded onto 96-well clear bottom tissue culture plates then treated with the indicated treatments at the indicated concentrations and durations. Following treatment, cells were incubated with the WST-1 reagent for 4 hours at 37°C with 5% CO2. The WST-1 reagent is cleaved to formazan by cellular enzymes in actively metabolizing cells. The formazan product was quantified by taking absorbance readings at 450 nm on a Wallac Victor³ 1420 Multilabel Counter (PerkinElmer, Woodbridge, ON, Canada). Cellular viability as a measure of metabolic activity was expressed as percentages of the solvent control groups.

The effect of SEE on the transcriptional activity of NF-κB and GRE was confirmed using the Cignal reporter assay kits (Qiagen) consisting of NF-κB-responsive firefly luciferase reporter (#CCS-013 L) and GR-responsive firefly luciferase reporter (#CCS-006 L), respectively, and a Renilla construct luciferase reporter (40:1). HepG2 cells (2 × 10⁵ cells/well) were seeded in 24-well culture plates, incubated for 24 h, and then transfected with 200 ng of plasmid DNA using 3 μL of Genjet™ in vitro DNA transfection reagent according to the manufacturer’s instructions.
After 18 h of transfection, the cells were treated with medium containing HC (1 μM) or SEE (1000 and 5000 μg/mL) for 30 min, followed by stimulation with 10 ng/mL TNF-α for 5 h for the NF-κB pathway analysis or medium containing 1 μM HC or SEE (1000, 2500, and 5000 μg/mL) for 6 h for the GRE pathway analysis. The expression of the luciferase reporter gene was analyzed using the DualGlo luciferase reporter system kit. Luminescence was measured using the Wallac Victor3 1420 multilabel counter (PerkinElmer, MA, USA). The level of firefly luciferase activity was normalized to that of Renilla luciferase in each experiment.

A WST-1 colorimetric assay was used to quantify cell viability of cells treated with curcumin and its analogs (compound A-J), following a previously published protocol [15 (link)]. Briefly, A375 and NHF cells were seeded in ninety-six well clear bottom tissue culture plates then treated with the indicated compounds for 48 h. The cells were then incubated with the WST-1 reagent (Roche Applied Sciences, Indianapolis, IN, USA) for 4 h at 37 °C, 5% CO2, and 95% humidity. The WST-1 reagent is cleaved by viable cells to formazan through its enzymes. Absorbance was read at 450 nm on a Wallac Victor3 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA) to determine cell viability.