The estimation of cell viability was performed by the MTT assay according to the manufacturer protocol (Sigma-Aldrich). First, cells were incubated with estrogens (E2, 2-MeOE2 and 16α-OHE1) or Cr(VI) independently to evaluate the cytotoxicity of this compounds to SKOV-3. The following concentrations of compounds were used—E2: 0.01, 0.1, 0.5, 1, 5, 10, 25, 50, 100 and 200 µM; 2-MeOE2 and 16α-OHE1: 0.001, 0.1, 1, 10 and 50 µM; Cr(VI): 0.1, 1, 5, 10 and 50 µM. Then, some of the concentrations were chosen for further studies of combined effect of E2 or its metabolites with Cr(VI) on the SKOV-3 line. Cells were seeded onto 96-well culture plates at a concentration of 5 × 104 cells/well (Nunc, Nunclon Surface, Biokom, Janki, Poland).
Nunclon surface
Manufactured by Thermo Fisher Scientific
Sourced in Finland
The Nunclon Surface is a type of cell culture surface treatment developed by Thermo Fisher Scientific. It is designed to promote cell attachment and growth for a variety of cell types in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Infinite 200, by Tecan (3 mentions)
Ultra amplex red hydrogen peroxide peroxidase assay kit, by Thermo Fisher Scientific (2 mentions)
Mtt assay, by Merck Group (1 mentions)
Trypsin, by Harvard Bioscience (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Harvard Bioscience (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Trypsin edta, by Harvard Bioscience (1 mentions)
Amplex ultrared hydrogen peroxide, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Sartorius (1 mentions)
Streptomycin, by Sartorius (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Sartorius (1 mentions)
L glutamine, by Sartorius (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Bhk 21, by American Type Culture Collection (1 mentions)
Fugene hd, by Promega (1 mentions)
8 protocols using nunclon surface
1
Cytotoxicity Assay for SKOV-3 Cells
2
Assessing Adhesion of Cryopreserved F. psychrophilum
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For study of the adhesion ability, the cryopreserved F. psychrophilum stocks were cultured for 5 days on TYES agar and recultivated another 3 days before inoculation of bacteria into sterile (autoclaved 121°C, 20 min) aquaculture tank water to an OD520nm of 1.0, corresponding to approximately 109 CFU ml−1 (Sundell et al., 2020 (link)). Aliquots of 100 μl were added in triplicate to wells of a flat‐bottomed 96‐well polystyrene microtiter plate (Nunclon ∆ Surface, Nunc) while sterile fresh water was used as a negative control. After static incubation at 15°C for 1 h, the contents were discarded. To remove non‐adherent cells, the wells were washed three times with sterile 0.5% NaCl and air‐dried. A 125 μl volume of a 0.1% CV solution was then added to each well and incubated at room temperature for 45 min. After discarding the contents, the plates were washed three times by submersion in a container of tap water and air‐dried. Then, 150 μl of 96% ethanol was added to each well and incubated at room temperature for 15 min. A 100 μl volume of the solubilized CV was then transferred to a flat bottomed microtiter plate and the absorbance was quantified in a microplate reader (Victor2, Wallac) at 595 nm. Each isolate was examined in triplicate and the experiment was repeated three times.
3
AKT Isoform Knockout in Colon Cancer Cells
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The colon cancer isogenic DLD-1 X-MAN™ cell lines were obtained from Horizon Discovery Ltd., (Cambridge, UK) with the different AKT isoforms genetically knocked out, cat. no. HD-R00-001, HD-R00-002 and HD-R00-003. The cells were cultured in 75-cm2 culture flasks (Nunclon surface; Nunc, Roskilde, Denmark) in McCoy's 5A medium (Flow Laboratories, Irvine, UK) with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, 100 IU/ml penicillin and 10 µg/ml streptomycin (Biochrom GmbH, Berlin, Germany). The cells were cultured in a humidified incubator with 5% CO2 at 37°C and trypsinized with trypsin-EDTA, 0.25% trypsin, 0.02% EDTA (Biochrom GmbH).
4
Fluorometric Quantification of Hydrogen Peroxide
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Hydrogen peroxide (H2O2) concentrations in aorta homogenates were determined by fluorescence in black microplates (Nunclon® Surface, Thermo Fisher Scientific®, Vantaa, Finland), using the Amplex® UltraRed Hydrogen Peroxide (10-acetyl-3,7-dihydroxiphenoxazine) Assay (Invitrogen®, Paisley, United Kingdom). This reagent, in the presence of peroxidase (horseredish peroxidase, HRP), stoichiometrically reacts with H2O2 to form a red-fluorescent oxidation product, resorufin. A standard curve of H2O2 was prepared by dilution into reaction buffer, with concentrations ranging from 0 to 10 μmol.L-1. Next, 50μL from the prepared curve points or from the aorta homogenate samples (or plasma) were plated in duplicate, with the addition of 50μL of working solution/HRP for beginning the reaction. The microplates were incubated at room temperature for 120 minutes, protected from light, and read at wave lengths of 530 nm and 590 nm for excitability and emission, respectively, in a microplate reader (Tecan 200 Infinite® 200 PRO plate reader, Männedorf, Switzerland).
5
BHK Cell Culture Maintenance
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BHK cells were BHK-21 (ATCC). BHK cells were grown and maintained according to standard protocols using DMEM, supplemented with 10% FBS (Biological Industries), 100 U/ml penicillin, 100 µg/ml streptomycin (Biological Industries), 2 mM l -glutamine (Biological Industries), 1 mM sodium pyruvate (Gibco), and 30 mM Hepes buffer, pH 7.3, at 37°C (Biological Industries). Cells were grown at 37°C with 5% CO2. Cells were transfected using a mixture of 8 µl Fugene HD (Promega) and 112 µl Opti-MEM (Gibco) with plasmids specified in the DNA constructs section in 1 ml for every 35-mm plate (Nunclon surface; Thermo Fisher Scientific) without replacing the medium (Avinoam et al., 2011 (link)).
6
Quantifying MPO Activity in Plasma
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Similarly to the determination of H2O2 levels, MPO activity in plasma samples was performed using the Ultra Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen, Paisley, UK), according to the manufacturer's instructions. A standard curve was prepared, with MPO concentrations ranging from 0.0312 to 1.0 UI·mL−1 (Sigma, St. Louis, USA). Then, 50 μL of the curve points or from the samples were plated, with the addition of the Amplex Red/H2O2 working solution to start the reaction. Next, samples were incubated at room temperature for 150 minutes, protected from light. Finally, fluorescence was measured in a spectrofluorometer (Tecan 200 Infinite, Männedorf, Switzerland), using black microplates (Nunclon Surface, Thermo Fisher Scientific, Vantaa, Finland) at the wavelengths of 530 and 590 nm for excitation and emission, respectively.
7
Plasma Nitrite Quantification via DAN Assay
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The quantification of plasma nitrite concentrations was performed based on the protocol described by Misko et al. [30 (link)], with adaptations for microplates. This fluorimetric assay is based on the reaction between nitrite and the compound 2,3-diaminonaphthalene (DAN), originating 2,3-diaminonaphthotriazole. Initially, plasma samples were filtered using a 10 dKa molecular weight filter (Millipore, Missouri, USA). Then, using black 96-well microplates (Nunclon Surface, Thermo Fisher Scientific, Vantaa, Finland), to 50 μL of each sample (in duplicate) were added 100 μL of deionized water. Next, 10 μL of DAN (0.05 mg·mL−1 in HCl 0.62 M) were added and mixed immediately, with DAN always protected from light. After incubation at 20°C for 10 minutes, the reaction was stopped by the addition of 5 μL of NaOH (2.8 M). The compound formed was quantified in a spectrofluorometer (Tecan 200 Infinite, Männedorf, Switzerland), at 365 nm and 410 nm for excitation and emission, respectively. Nitrite concentrations were calculated based on a standard curve of nitrite.
8
Plasma H2O2 Quantification by Fluorescence
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The quantification of plasma H2O2 levels was performed by fluorescence (Tecan 200 Infinite, Männedorf, Switzerland), with a commercial kit (Ultra Amplex Red Hydrogen Peroxide/Peroxidase Assay kit, Invitrogen, Paisley, UK), according to the manufacturer's instructions. In the presence of peroxidase (horseredish peroxidase, HRP), the Amplex Red reagent stoichiometrically reacts with H2O2 to form a red-fluorescent oxidation product, resorufin. A standard curve of H2O2 was prepared, with concentrations ranging from 0 to 10 μM. Next, 50 μL from the curve points or from the samples were plated in duplicate, with the addition of 50 μL of the reagent/HRP solution to start the reaction. Finally, the black microplates (Nunclon Surface, Thermo Fisher Scientific, Vantaa, Finland) were incubated at room temperature for 120 minutes, protected from light and read at wavelengths of 530 and 590 nm, respectively, related to excitation and emission.
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