1 × 10 [4 (link)] HUVEC or hBMECs were cultured in triplicate in Nunc™ cell culture treated 96-well plates for 24 h. Aβ1-42, scrambled control peptide DMSO or NOX inhibitors were added as indicated for 4 h 200 μM 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), 5 μM diethyldithiocarbamate (DETC) and 25 μM deferoxamine were then added to the cultures for 45 min 50 μL of supernatant were transferred into the Hirschmann precision micropipettes and read using an e-scan (Noxygen, Germany), as previously described [33 (link)]. EPR spectra were recorded using the following EPR settings: centre field 3492.5 G, field sweep 60 G, modulation amplitude 2 G, sweep time 10 s, number of scans 10, microwave frequency 9.39 GHz, microwave power 20 mW, conversion time 327.68 ms, time constant 5242.88 ms. A calibration curve was obtained from standard CM● diluted to concentrations of 0, 0.3, 1, 3, 10, and 30 μM and utilised to estimate the CM● concentration in the samples as described in Supplementary Fig. 1 . The CMH oxidation rate was obtained using the formula below:
Nunc cell culture
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nunc Cell Culture is a range of lab equipment designed for cell culture applications. It provides a controlled environment for the growth and maintenance of cells in vitro. The products include cell culture flasks, plates, dishes, and other consumables.
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Lab products found in correlation
8 protocols using nunc cell culture
1
Quantification of Oxidative Stress in HUVEC Cells
2
Cell Culture Reagents and Supplies
3
Cytotoxicity Assay for NK Cell Activity
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The cytotoxicity of NK cells against target cells was assessed by a fluorometric cytotoxicity assay (20 (link)). Target cells are stained with 30 mM calcein-AM (Molecular Probes, Thermo Fisher Scientific) for 1 h at 37°C. NK cells and labeled target cells were co-cultured in U-bottom 96-well plates (Nunc cell culture, Thermo Fisher Scientific) in triplicate at effector:target (E:T) ratios of 0.1:1 to 30:1 per 1×104 target cells at 37°C and 5% CO2 for 4 h. To assess the ADCC of NK cells, 0.5 ng/mL of rituximab (Roche, Basel, Switzerland) was added. RPMI1640 medium containing 10% FBS or 0.1% triton-X100 (Sigma-Aldrich, St. Louis, MO, USA) was added to the target cells as spontaneous and maximum release controls, respectively. Measurements were made at an excitation wavelength of 485 nm and emission wavelength of 535 nm using a fluorometer (VICTOR 3; PerkinElmer, Waltham, MA, USA). The percentage of specific calcein-AM release was calculated as follows:
4
In Vitro Maturation and Fertilization of Sheep Oocytes
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Groups of 20–25 COCs were matured in 600 μL of TCM 199 supplemented with heat-treated oestrous sheep serum (10%, OSS), pyruvate (0.36 μmol/L), cysteamine (100 μmol/L), Follicle-stimulating hormone (FSH; 1 IU/mL), Luteinizing hormone (LH; 1 IU/mL) under mineral oil, in four-well dishes (Nunc Cell Culture, Thermo Fisher Scientific, Waltham, Massachusetts, USA) in a humidified atmosphere of 5% CO2, at 38.5 °C for 24 h [33 (link)]. After IVM, COCs were completely denuded of granulosa cells via gentle pipetting with a fine bore glass pipette. Oocytes at the metaphase II (MII) stage were selected under a stereomicroscope (Olympus SZ-PT, Italy) for the presence of the first polar body and randomly assigned to the analyses. For IVF groups of MII oocytes from the different experimental groups were co-incubated with frozen-thawed ram spermatozoa (1 × 106 spermatozoa/mL), selected by the swim-up technique in synthetic oviductal fluid (SOF, [34 (link)]) supplemented with OSS (2%), heparin (1 μg/mL), hypotaurine (1 μg/mL) for 16 h in a humidified atmosphere of 5% CO2 at 38.5 °C [33 (link)].
5
Progerin-Induced NDF Migration Assay
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Ibidi cell culture inserts (Ibidi Cat. # 80209) were mounted on 24‐mm‐diameter coverslips (Nunc Cell Culture, Thermo Fisher). NDF harbouring doxycycline‐inducible pTRIPZ‐v5‐progerin were grown to confluence within culture inserts. NDF were induced ± 1 μg/ml doxycycline for 3 days and culture inserts removed to initiate cell proliferation and migration at the edge. Cells were fixed 72 hr postinsert removal and analysed by immunofluorescence microscopy.
6
Quantitative ELISA for RBD/HA Antibodies
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One hundred microliters of RBD or influenza A H1N1 HA recombinant protein (4 μg/ml; A42579, Thermo Fisher Scientific) was added into micro titer plates (Nunc Cell Culture, Thermo Fisher Scientific) for overnight at 4°C. After that, 200 μl of 5% (w/v) bovine serum albumin (B6717, Sigma-Aldrich) in phosphate-buffered saline with 0.1% Tween 20 (PBS-T) buffer was added to each well to block nonspecific binding and incubated at 37°C for 1 hour. After three times of wash with PBS-T, serum samples with serial dilutions (1:100, 1:1000, 1:3000, 1:5000, 1:10,000, 1:50,000, 1:100,000, and 1:200,000) and control serum samples (1:100 dilution) were added to the wells and incubated at 37°C for 1.5 hours. After washing intensively, 100 μl of horseradish peroxidase–labeled anti-mouse IgG secondary antibody with 1:10,000 dilution was added for 1 hour of incubation at 37°C. After three times of wash, 100 μl of trimethylboron was added into each well and incubated at room temperature for 30 min. Fifty microliters of 2 M H2SO4 (Sigma-Aldrich) was used for stopping the reaction, and the optical absorption was determined at 450 nm via a plate reader. The endpoint IgG titer was determined as the highest reciprocal dilution of serum that exhibits an optical density greater than twofold of the mean control group.
7
In Vitro Oocyte Maturation Protocol
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Ovaries from control and stored groups were sliced with a scalpel blade to release the cumulus-oocyte complexes (COCs). The COCs were collected in sterile Petri dishes in dissection medium (DM; 25 mM Hepes-buffered TCM 199) supplemented with 0.1% (w/v) polyvinyl alcohol (PVA) and antibiotics (100 μg/ml penicillin and streptomycin). Only the oocytes with darkly pigmented ooplasm and completely surrounded by at least one layer of cumulus cells were selected for in vitro maturation (IVM). The COCs were matured in groups of 25–35 in 650 μl of TCM 199 supplemented 0.36 mM pyruvate, 2 mM glutamine, 2.2 mM calcium lactate, 1.2 mM cysteine, 4 mg/ml BSA fatty acid free and FSH 1 IU / ml and LH 1 IU / ml (Pluset; Bio98, Milan, Italy) under mineral oil, in 4-well dishes (Nunc Cell Culture, Thermo Fisher Scientific, Waltham, Massachusetts, USA) in a humidified atmosphere of 5% CO2, at 38.5 °C for 24 h [46 (link)].
8
Exosome Isolation from Cell Cultures
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Exosomes were prepared from conditioned media from cell cultures, using Total Exosome Isolation (TEI) reagent (for cell culture media) from Invitrogen, Life Technologies, in accordance with the manufacturer's instructions. Conditioned media were centrifuged at 2000g for 30 min, 4 °C to remove cells and cell debris and the resulting supernatants were mixed with 0.5 volumes of TEI reagent and centrifuged at 10,000g, 4 °C, 1 h, following overnight 4 °C incubation. Exosome pellets were twice washed by re-suspension in ice-cold PBS followed by centrifugation at 100,000g, 1 h, 4 °C (Gardiner et al., 2016 ). Unused intact exosomes were stored at − 80 °C as PBS suspensions. For F9-RARE-lacZ RA reporter cells experiments, a pool of exosomes isolated from a 75 cm2 Thermo Scientific™ Nunc™ Cell Culture treated flasks with Filter Caps of 99% confluent mouse cortical neurons, or astrocytes or NG2 + cells were used.
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