The level of nitrite, which is the stable reaction product generated from NO with molecular oxygen, was used as an indicator of NO production. Briefly, NHDF cells in each well were treated with 55 mJ of UV, and subsequently with MED or CPO for 24 h, after which the supernatants were collected. For skin tissue, homogenates were collected and kept at −80 °C until use. Duplicates of 100 μL supernatant of homogenate or culture media were added to 96-well plates and mixed with 100 μL modified Griess reagent (Invitrogen, California, USA). The absorbance of each well was measured at 540 nm using a Versa max plate reader (Molecular Devices). A standard curve with increasing concentrations of sodium nitrite was generated in parallel and used for quantification.
Modified griess reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Modified Griess reagent is a chemical compound used in analytical procedures. It is primarily employed for the detection and quantification of nitrite ions in various sample types. The reagent facilitates a colorimetric reaction that produces a colored complex, allowing for the measurement of nitrite concentrations.
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4 protocols using modified griess reagent
1
Nitrite Assay for NO Production
2
Nitrite Levels in ARPE19 Cells
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The level of nitrite in the ARPE19 cells was determined as an indicator of NO production using the Griess reagent as in a previous study [22 (link)]. Briefly, the culture supernatants were collected from subset groups of ARPE19 cells after subsequent treatment with MED, A2E, or BL. These supernatants (100 μL) were mixed with 100 μL of modified Griess reagent (Invitrogen Co., Carlsbad, CA, USA) in 96-well plates. After incubation for 5 min, the absorbance at 540 nm was detected using a VersamaxTM microplate reader (Molecular Devices, San Jose, CA, USA).
3
Quantification of Nitric Oxide Levels
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The level of nitrite, which is the stable reaction product generated from NO with molecular oxygen, was used to indicate NO production. The mid colon tissue (20 mg) from mice (n = 5) in subset group was homogenized in ice-cold 1× PBS (pH 7.4) and centrifuged at 14,000 rpm for 5 min at 4 °C. The supernatants of the tissues were collected from mid colon and kept at −80 °C until use. Duplicates of 100 μL of the supernatant of homogenate were added to 96-well plates and mixed with 100 μL modified Griess reagent (Invitrogen, Carlsbad, CA, USA). The absorbance of each well was measured at 540 nm using a Versa max plate reader (Molecular Devices). A standard curve with increasing sodium nitrite concentrations was generated in parallel and used for quantification.
4
Quantifying Nitrite Levels in Macrophages
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The level of nitrite, a stable reaction product generated from NO with molecular oxygen, was used as an indicator of NO production. The NO concentration in the culture supernatant of RAW264.7 macrophages was measured using Griess reagent (Invitrogen Co., Carlsbad, CA, USA) as described previously [50 (link)]. Briefly, RAW264.7 macrophages were preincubated with LPS (1 μg/mL) for 2 h and then treated with 1× PBS or AA (10, 20, and 40 μM) for 12 h. After collecting the supernatants, 100 μL of each was mixed with 100 μL of modified Griess reagent (Invitrogen Co.) in 96-well plates and incubated for 5 min. The absorbance of each well was measured at 540 nm using a VersamaxTM microplate reader (Molecular Devices). A standard curve with increasing concentrations of sodium nitrite was generated in parallel and used for quantification.
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