Purified monocytes were co-cultured for 40 min with E. coli at a monocyte: E. coli ratio of 1:100. The cell suspension was then mixed with Diogenes reagent (National Diagnostics, USA) according to the manufacturer's instructions and superoxide was quantified by recording luminescence (relative light units) using a luminometer (FLUOstar OPTIMA, BMG Labtech) according to the manufacturer's instructions.
Diogenes reagent
Manufactured by National Diagnostics
Sourced in United States
Diogenes reagent is a chemical compound used in laboratory analysis. It is a colorimetric reagent that is used to detect the presence of certain substances in a sample. The core function of Diogenes reagent is to facilitate qualitative and quantitative analysis through color-based detection.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar optima, by BMG Labtech (1 mentions)
Dpi chloride, by Merck Group (1 mentions)
Luminoskan luminometer, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution buffer, by Thermo Fisher Scientific (1 mentions)
Fura 2am dye, by Thermo Fisher Scientific (1 mentions)
Clariostar plus microplate reader, by BMG Labtech (1 mentions)
4 protocols using diogenes reagent
1
Monocyte Superoxide Production Assay
2
Measuring Oxidant Production in HL-60 Cells
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Differentiated HL-60 cells were collected and washed twice with Hank's balanced salt solution (HBSS; Invitrogen). Cells (2×106/mL, 50 µL) were treated for 15 minutes at 37°C with or without 10 µM DPI chloride (Sigma-Aldrich). Cells were stimulated with 1 µM fMLP. Extracellular O2− production was measured as SOD-inhibitable chemiluminescence detected using Diogenes reagent (National Diagnostics, Atlanta, GA, USA) using a 96-well plate Luminoskan luminometer (Thermo, Waltham, MA, USA). Extracellular H2O2 was measured by a luminol/HRP-based chemiluminescence assay. Briefly, cells (2×106/mL, 50 µL) were collected as above and treated with or without 10 µM DPI (37°C for ten minutes). An equal volume of HBSS containing 1 mM luminol and 20 U/mL HRP was added following DPI treatment. Luminescence was estimated as above. Superoxide production and H2O2 production was presented as integrated luminescence value (relative light unit) [70] (link).
3
Kinetic ROS detection by chemiluminescence
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Kinetic ROS detection measurements were performed by chemiluminescence in 96-well plates at 37 °C over a 45-min time course using a Luminoskan luminometer (Dharmacon) as previously described (Boudreau et al, 2009 (link)). Briefly, ∼2.5 × 104 cells were collected by trypsinisation and washed twice with Hank's balanced salt solution buffer (Invitrogen) by centrifugation. SOD3-sensitive superoxide production was measured using Diogenes reagent (National Diagnostics, Atlanta, GA, USA). Extracellular H2O2 was measured by a luminol/HRP-based chemiluminescence assay.
4
Microplate-based Superoxide and Calcium Signaling
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All measurements were performed with a CLARIOstar Plus Microplate Reader (BMG LABTECH) at 37 °C. Black chimney, clear 96-well plates (Greiner 655090) were used for Ca2+ measurements, and white chimney, solid bottom, 96-well plates (Greiner 655083) were used for superoxide measurements. The stimuli were injected or pipetted into the wells, and the final volume was 100 μl/well in all cases.
Superoxide measurements were performed using circa 40,000 cells/well in suspension in a 1:1 mixture of H-medium (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.8 mM CaCl2, 5 mM glucose, 10 mM HEPES) and Diogenes reagent (National Diagnostics). The extracellular superoxide signal was detected in luminescence mode.
Ca2+ measurements were performed on confluent, adherent cell cultures in H-medium. The cells were loaded with 2 μM Fura-2 AM dye (Life Technologies) for 30 min at 37 °C in the dark, then washed with H-medium. Fura-2 was excited at 335 and 380 nm, and respective emission signals were detected at 510 nm, and their ratio (335/380) was analyzed.
Superoxide measurements were performed using circa 40,000 cells/well in suspension in a 1:1 mixture of H-medium (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.8 mM CaCl2, 5 mM glucose, 10 mM HEPES) and Diogenes reagent (National Diagnostics). The extracellular superoxide signal was detected in luminescence mode.
Ca2+ measurements were performed on confluent, adherent cell cultures in H-medium. The cells were loaded with 2 μM Fura-2 AM dye (Life Technologies) for 30 min at 37 °C in the dark, then washed with H-medium. Fura-2 was excited at 335 and 380 nm, and respective emission signals were detected at 510 nm, and their ratio (335/380) was analyzed.
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