The effect of 3 and 27 on biofilm formation was evaluated through quantification of total biomass by crystal violet staining [26 (link),39 (link)]. Briefly, each compound, in concentrations ranging between 128 and 16 μg/mL, was added to C. albicans ATCC 10,231 suspensions prepared in RPMI-1640 broth medium supplemented with MOPS, from overnight SDA cultures, at a final concentration of (1.0 ± 0.2) × 106 CFU/mL, as determined by cell counts using a hemocytometer. A control with appropriate concentration of DMSO, as well as a negative control (RPMI-1640 alone), was included. Sterile 96-well flat-bottomed untreated polystyrene microtiter plates were used. After a 48-h incubation at 37 °C, the biofilms were stained with 1% (v/v) crystal violet (Química Clínica Aplicada, Amposta, Spain) for 5 min. The stain was solubilized with 33% (v/v) acetic acid (Acetic acid 100%, AppliChem, Darmstadt, Germany), and the biofilm biomass was quantified by measuring the absorbance of each sample at 570 nm in a microplate reader (Thermo Scientific Multiskan® EX, Thermo Fisher Scientific, Waltham, MA, USA). The background absorbance (RPMI-1640 without inoculum) was subtracted from the absorbance of each sample and the data are presented as percentage of control. Three independent assays were performed in triplicate for each experimental condition.
Thermo scientific multiskan ex
Manufactured by Thermo Fisher Scientific
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The Thermo Scientific Multiskan EX is a microplate reader designed for absorbance-based measurements. It is capable of performing photometric measurements in 96-well and 384-well microplates. The device can be used for a variety of applications, such as enzyme-linked immunosorbent assays (ELISA), cell-based assays, and other absorbance-based analyses.
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11 protocols using thermo scientific multiskan ex
1
Evaluating Biofilm Formation: 3 and 27
2
Etanercept Pharmacokinetic Profiles Comparison
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In order to characterize differences in etanercept delivery by the different routes of administration, we used normal rats to determine the PK profiles following a single dose of 1 mg etanercept in 100 uL delivered via SOFUSA™ (n = 6), conventional IV (in the tail vein, n = 6), SC (dorsolateral injection at the same site as the SOFUSA™ application, n = 4), or ID injections (symmetrical dorsolateral injections 2 × 50 uL for a total dose of 1 mg etanercept, n = 6). Other than the SOFUSA™ delivery, all administration was conducted using a 31-gauge needle. At 2, 4, 8, 12, 24, and 36 h after administration, animals were anesthetized under isoflurane, and 200 μL of blood was drawn from the jugular vein. The etanercept concentration in serum was quantified using the Etanercept ELISA Kit (ABIN: 1540251) (Matriks Biotechnology Co., LTD., Ankara, Turkey). Optical density was measured at 450 nm using Thermo Scientific Multiskan EX (Thermo Fisher Scientific, Waltham, MA, USA).
3
Cell Growth and Metabolic Activity Assays
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Cell growth was assessed using the trypan blue exclusion assay. Control and treated cells were plated in duplicates in 24-well plates. At pre-determined time points, MSCs were released from the plate using 0.05% Trypsin-EDTA (R-001-100, Life Technologies, California, USA), then equal volumes of cells and trypan blue dye and counted using a haemocytometer.
In parallel, the MTT assay was performed to evaluate the metabolic activity of MSCs. Control and treated cells were seeded in duplicates onto flat-bottom 96-well plates. At each time point, cells in 90 µl media were incubated with 10 µl of a 5 mg/ml MTT substrate solution, thiazolyl blue tetrazolium bromide salt (Sigma-Aldrich Co, Missouri, USA) for 4 hours. The reaction was stopped by the addition of 100 µl of solubilizing solution (12 mM HCl, 346 mM SDS and 5% isobutanol); formazan dye was then quantified at a wavelength of 595 nm using a scanning multi-well spectrophotometer (Thermo Scientific Multiskan EX, Thermo Scientific, USA). Proliferation results were reported as percentages of control.
In parallel, the MTT assay was performed to evaluate the metabolic activity of MSCs. Control and treated cells were seeded in duplicates onto flat-bottom 96-well plates. At each time point, cells in 90 µl media were incubated with 10 µl of a 5 mg/ml MTT substrate solution, thiazolyl blue tetrazolium bromide salt (Sigma-Aldrich Co, Missouri, USA) for 4 hours. The reaction was stopped by the addition of 100 µl of solubilizing solution (12 mM HCl, 346 mM SDS and 5% isobutanol); formazan dye was then quantified at a wavelength of 595 nm using a scanning multi-well spectrophotometer (Thermo Scientific Multiskan EX, Thermo Scientific, USA). Proliferation results were reported as percentages of control.
4
Cell Viability Assay for Cancer and Normal Cells
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Cells were seeded (5000 cells/well) in sterile 96-well plates in complete growth medium. They were incubated overnight to allow cell attachment. Following overnight incubation, the HCT-116 cells were treated with 3 to 20 mg/mL, LoVo cells were treated with 5 to 60 mg/mL andHDF cells were treated with 3 to 50 mg/mL concentrations of STH and MH, while control wells were treated only with medium. After 24, 48 and 72 h incubation, 30 μL of RPMI medium containing 2 mg/mL of MTT was added and cells were incubated for other 4 h. The generated formazan crystals were solubilized by adding 100 μL of DMSO, and quantified by a microplate reader (ThermoScientific Multiskan EX, Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 590 nm. The percentage of viable was calculated as (absorbance of treated cells/absorbance of control cells) × 100.
5
Quantifying Murine GROα in Senescent Cells
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The protein levels of murine GROα were assessed in the supernatants of proliferating and senescent TC-1 and TRAMP-C2 cells using high-sensitivity ELISA kits (cat. no. DY453; R&D Systems, Inc.). After 4-day induction of senescence, cells were washed twice with PBS to remove DTX in the medium to avoid its possible influence on the results. K1836 was thereafter added for 72 h with repeated supplementation every 24 h. After cell treatment at 37°C, the medium was changed and the cells were cultivated for another 48 h in fresh medium. Experiments were performed according to the manufacturer's protocol and absorbance was quantified using a Thermo Scientific Multiskan EX (Thermo Fisher Scientific, Inc.).
6
Bilberry's Effect on Cell Proliferation
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Cell proliferation was measured with the commercial Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, cells were cultured in 96-well plates at a density of 5 × 103 cells in normal culture medium (100 μL) for 24 h, after which the culture medium was changed and the cells exposed to 0.2, 1, 5, 10, and 25 mg/mL of bilberry. PBS (25 µL/100 µL) was used as a control. After 24- and 72-h incubations, the cells were labelled using 10 μM BrdU per well and re-incubated for 2 h at 37 °C in a humidified atmosphere. Culture medium was removed, cells were fixed, and DNA was denatured in one step by adding FixDenat (Roche Diagnostics, Basel, Switzerland). Next, the cells were incubated with anti-BrdU-POD for 90 min at room temperature. After removal of the antibody conjugate, cells were washed twice, and substrate solution was added. The reaction product was quantified by measuring absorbance using a scanning multi-well spectrophotometer (Thermo Scientific Multiskan EX, Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm with a reference wavelength of 690 nm. There were two (HSC-3 cells) or three (HMK and IHGK cells) independent experiments with six replicates in each assay.
7
Colorimetric Assay of COX Inhibitors
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This assay examined the inhibitory potency of the target structures towardboth COX isoforms via colorimetric screening assay kit (Cayman Chemical, MI, United States) based on the directions provided by the manufacturer. ELISA reader Thermo Scientific Multiskan® EX (Thermo scientific, United States) was used. IC50 (concentration at which there was 50% inhibition) values were calculated using GraphPad Prism 8 analysis software (Graph-Pad, San Diego, CA, United States) to form the dose–response curves of eight concentrations of each test compound. Serial dilutions of CLX, as a reference standard, and tested compounds at concentrations of 1000, 300, 100, 30, 10, 3, 1, and 0.30 nM were used in the assay. IC50 of the tested compounds were determined using three different experiments. The selectivity (SI) of each ompound was measured using the COX-2/COX-1 ratio, which compares the IC50 of COX-2 and COX-1.
8
Evaluating Antibiotic Antibiofilm Activity
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To evaluate the antibiofilm activity, suspensions of GO and GO-AZ were drop-casted on a 96-well flat bottom plate by slowly drying in an air oven at 50 °C [25 (link)]. The coated wells’ biofilm inhibition efficiency was analyzed by static biofilm forming assays of C. albicans and was performed on 96-well polystyrene plates (SPL Life Sciences, Korea) [26 (link)]. Briefly, stationary phase cells were cultured with or without samples at varying concentrations in PDB medium without shaking for 24 h at 37 °C. After incubation, the cell growth was measured at 620 nm and the supernatant was discarded and washed three times with distilled water to remove non-adherent cells. Excess water was removed, and the plates were dried before staining with 300 μL of crystal violet (0.1%, v/v) for 20 min at room temperature. Stained plates were washed, and the adsorbed crystal violet was dissolved after adding 95% ethanol for 15 min. The formed biofilm was measured at 570 nm using a Thermo Scientific Multiskan EX (Thermo Fisher Scientific, Vantaa, Finland). Biofilm inhibition was quantified from six replicates, and the results are presented as the averages with standard deviations.
9
Biofilm Formation Inhibition Assay
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For compounds with antibacterial activity, their effect on biofilm formation was evaluated using the crystal violet method. Briefly, bacterial suspensions of 1 × 106 CFU mL−1 were prepared in Tryptone Soy broth (TSB-Biokar Diagnostics, Allone, Beauvais, France) supplemented with 1% (p/v) glucose (d (+)-glucose anhydrous for molecular biology, PanReac AppliChem, Barcelona, Spain); and four concentrations of compound were tested: 2 × MIC, MIC, ½ MIC and ¼ MIC, keeping final in-test concentration of DMSO below 1%. A control with inoculum and culture media, a control with appropriate concentration of DMSO, as well as a negative control (TSB alone) were included. Sterile 96-well flat-bottomed untreated polystyrene microtiter plates were used. After a 24 h incubation at 37 °C, the biofilms were heat-fixed for 1 h at 60 °C and stained with 0.5% (v/v) crystal violet (Química Clínica Aplicada, Amposta, Spain) for 5 min. The stain was resolubilized with 33% (v/v) acetic acid (acetic acid 100%, AppliChem, Darmstadt, Germany) and the biofilm biomass was quantified by measuring the absorbance of each sample at 570 nm in a microplate reader (Thermo Scientific Multiskan® EX, Thermo Fisher Scientific, Waltham, MA, USA).40,43 (link) Three independent experiments were performed, in triplicate.
10
DPPH Radical Scavenging Assay
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One hundred microliters of DPPH 0.20 mM in absolute methanol and 100 μL of the appropriate compound (0.32, 0.16, 0.08, 0.04, 0.02, 0.01 mM final concentrations) dissolved in DMSO were poured into a 96-well plate. The mixtures were incubated for 30 min at room temperature and protected from light. The absorbance was recorded at 517 nm in a transparent 96-well test microplate (Multiskan-EX Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA). The results are expressed as percentage of DPPH radical present for each concentration of derivatives. To determine the antioxidant activity of each compound, the percentage of the DPPH radical-scavenging activity was calculated by the following equation: [1 − (A1 − A2)/(ADPPH − AS)] × 100, where: A1 = Absorbance of the compound with DPPH, A2 = Absorbance of the compound, ADPPH = Absorbance of DPPH (diluted 1:1 with solvent) and AS = Absorbance of DMSO.
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