Activation of T lymphocytes was evaluated by determining the concentration of IFNγ in the clarified supernatants from cultured T lymphocytes using a commercial ELISA specific for ch-IFNγ (Invitrogen, La Jolla, CA, Pei et al., 2003 (link), Singh et al., 2010a (link), Singh et al., 2010b (link)). Nitric oxide secretions induced in the cultured macrophage and T lymphocyte combinations were quantified using a Griess reagent assay according to the manufacturer's protocol (Sigma-Aldrich, St. Louis, MO) and described by Singh et al. (Singh et al., 2010a (link), Dawes et al., 2014 (link)). The concentration of nitrite produced was determined using sodium nitrite solutions with concentrations of 1–20 μm as standards. The concentration of any non-specific production of nitric oxide by soluble factors was adjusted by subtracting the nitrite concentration of supernatants from macrophages cultured without T lymphocytes from the supernatants of the macrophages cultured with T lymphocytes.
Griess reagent assay
Manufactured by Merck Group
Sourced in United States
The Griess reagent assay is a colorimetric method used to detect and quantify nitrite ions in various samples. The assay utilizes a chemical reaction involving the Griess reagent, which produces a colored azo compound in the presence of nitrite. This reaction allows for the spectrophotometric measurement of nitrite concentrations.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using griess reagent assay
1
Quantification of T Cell Activation
2
Nitric Oxide Quantification in Wnt3a-Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW264.7 cells were exposed to Wnt3a-CM or Ctl-CM, and the supernatant from each sample was collected at 12 and 24 h following PA challenge by centrifugation at 1,000 × g for 10 min at 4°C. NO levels were determined by measuring the levels of the stable end product, nitrite, using the Griess reagent assay (Sigma-Aldrich), according to the manufacturer's protocol. Briefly, 50 µl culture supernatant mixed with 50 µl sulfanilamide solution was added to wells in duplicate, and incubated for 10 min at room temperature in the dark. Subsequently, 50 µl N-1-napthylethylenediamine dihydrochloride solution was added to the wells and incubated for 10 min at room temperature in the dark. Absorbance at 540 nm was measured using the iMarkTM Microplate Absorbance Reader (Bio-Rad Laboratories, Inc.).
3
Nitrite Quantification in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amount of nitrite in cell lysates was measured through the Griess reagent assay (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturers’ instructions. Briefly, cells were resuspended in ice cold Nitrite Assay Buffer and chilled on ice for 10 min. Samples were centrifuged at 10,000× g at 4 °C for 5 min and the supernatants transferred to fresh tubes. A total of 10–100 μL of sample per well was added and the volume was adjusted to 100 μL with Nitrite Assay Buffer. Nitrite levels were then detected by measuring absorbance at 540 nm with an iMark microplate absorbance reader (BioRad, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!