Nitric oxide production was evaluated by the accumulation of nitrite, which is a stable break down product of NO. Levels of nitrite were assessed using the Griess reaction, which quantifies nitrite through the formation of a red azo dye. Cell culture supernatants (50 μl) were added to a 96-well plate and mixed with 50 μl of ice-cold sulfanilamide (2 mM) in hydrochloric acid (1.2 M). The mixture was incubated for 10 minutes at room temperature, protected from light. Then N-1-(1naphtyl)ethylenediamine (3 mM, 50 μl) was added followed by another incubation for 10 minutes at room temperature, protected from light. The absorbance of the resulting reaction solution was measured at 550 ± 10 nm wavelength in a Fluorstar Optima plate reader (BMG Labtechnologies) with wavelength correction at 380 ± 10 nm.
Fluorstar optima plate reader
Manufactured by BMG Labtech
Sourced in United States
The Fluorstar Optima is a highly sensitive plate reader designed for fluorescence-based assays. It offers fast, accurate, and reliable measurements across a wide range of samples and applications. The Fluorstar Optima is capable of reading microplates and supports various fluorescence detection modes.
Automatically generated - may contain errors
6 protocols using fluorstar optima plate reader
1
Nitric Oxide Quantification by Griess Reaction
2
Microglial H2O2 Production Assay
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H 2 O 2 production by microglia was measured in a fluorometric Amplex Red hydrogen peroxide/peroxidase assay. Isolated microglia were suspended in HBSS supplemented with 50 mM D-glucose. The reaction mixture of a control sample contained 6×10 4 microglia cells in black 96-well plate, 15 μM Amplex Red and 30 μg/ml horseradish peroxidase. Production of reactive oxygen species in microglial cells was induced by the treatment with PMA (30 nM). PKC inhibitors Gö6976
(1 μM) and Gö6983 (1 μM) were added together with the stimulant. The rate of H 2 O 2 production was assessed for two hours by continuously measuring fluorescence development in a Fluorstar Optima plate reader (BMG Labtechnologies) with excitation at 550 ± 10 nm and emission at 590 ± 10 nm.
(1 μM) and Gö6983 (1 μM) were added together with the stimulant. The rate of H 2 O 2 production was assessed for two hours by continuously measuring fluorescence development in a Fluorstar Optima plate reader (BMG Labtechnologies) with excitation at 550 ± 10 nm and emission at 590 ± 10 nm.
3
DICER1 Promoter Regulation in Hypoxia
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A 2.5 kb fragment from the 5′ flanking region of the DICER1 gene was previously described15 (link). MCF7 cells were transiently co-transfected with the DICER or CA9 promoter69 (link) constructs and pcDNALacZ using Lipofectamine (Invitrogen). Transfected cells were subcultured 16 hrs post-transfection, exposed to hypoxia and finally harvested 48 hrs after transfection. Luciferase and β-galactosidase activity was measured using a commercial kit (Applied Biosystems) and measured on the Fluorstar Optima plate reader (BMG Labtech).
4
DICER1 Promoter Regulation in Hypoxia
5
Macrophage-Cryptococcus neoformans Assay
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The macrophage-C. neoformans co-incubation assay was adapted from [23 (link)]. J774A.1 macrophage-like murine cells grown in DMEM (Dulbecco’s Modified Eagle Medium, Sigma-Aldrich) were harvested, washed in PBS, transferred to 96-well tissue culture plates at 105 cells/well, and incubated overnight at 37 °C and 5% CO2. The C. neoformans strain H99 was incubated in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) overnight with shaking at 37 °C, washed with PBS, and resuspended in DMEM medium at 103 cells/ml. Sphaerostilbellins (1 and 2 ) were prepared in serial 2-fold dilutions in DMEM and DMEM containing H99 cells to achieve a dose range of 0.03–32 µM. The macrophage medium was replaced with the DMEM medium containing compound or fungal cells plus compound and incubated for 24 to 48 h at 37 °C and 5% CO2. Macrophage viability was assessed by the addition of 10% alamarBlue and incubating for 3 h at 37 °C and 5% CO2 prior to fluorescence measurement (FLUORStar Optima plate reader, BMG Labtech, Cary, NC, USA). Fungal growth was assessed visually at 48 h. To test for fungal survival, samples from each well were plated onto YPD agar and incubated at 30 °C. Each compound was tested in duplicate biological replicates.
6
Mouse IP-10 ELISA Quantification
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