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Griess assay

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The Griess assay is a colorimetric method used to quantify nitrite (NO2-) levels in various samples. It is a simple and reliable analytical technique that measures the concentration of nitrite by forming a colored azo compound.

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Lab products found in correlation

Amplex red assay, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) F4 80 bm8, by BioLegend (1 mentions) Cytometric bead array, by BD (1 mentions) Fc blocking reagent, by BioLegend (1 mentions) Cd45 30 f11, by BioLegend (1 mentions) Gibco rpmi 1640 glutamax, by Thermo Fisher Scientific (1 mentions) Multiskan go spectrophotometer, by MultiSciences Biotech (1 mentions) Amplex ultrared assay, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade, by Thermo Fisher Scientific (1 mentions) Daf fm diacetate, by Thermo Fisher Scientific (1 mentions) Amplex ultrared, by Thermo Fisher Scientific (1 mentions) Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions) Ifn γ, by Cell Signaling Technology (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions) Alexa fluor 555 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)

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Griess assay kit Griess Reagent Assay kit Griess reagent assay

12 protocols using griess assay

1

Multiparametric Flow Cytometry of Liver Immune Cells

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For flow cytometry, single-cell suspensions of NPC and PBMC were incubated with Fc blocking reagent (Biolegend, San Diego, CA) for 10 min followed by a 30 min incubation with fluorescently-conjugated mAbs directed against mouse Mincle (4A9; MBL International, Woburn, MA), CD45 (30-F11), Gr1 (RB6-8C5), CD11b (M1/70), CD11c (N418), MHC II (M5/114.15.2), CD146 (ME-9F1), CD45.2 (30-F11), CD45.1 (A20), CD3 (17A2), CD4 (GK1.5), or F4/80 (BM8) (all Biolegend, San Diego, CA). For intracellular staining, cells were incubated for 4–6 hours with Brefeldin A (1:1000) before permeabilization of cells, and staining with fluorescently conjugated p-Syk (moch1ct) and iNOS (CXFNT) (all eBioscience, San Diego, CA). Human liver NPC and PBMC were stained with mAbs directed against CD45 (HI30), CD15 (W6D3), Lin (CD3/14/19/20/56), HLA-DR (L243; all Biolegend), or Mincle (polyclonal; Abcam, Cambridge, MA). Experiments were performed using the LSRII (BD Biosciences, Franklin Lakes, NJ) and analyzed using FlowJo software (Tree Star, Ashland, OR). Serum cytokine levels were determined using a cytometric bead array according to the manufacturer’s protocol (BD Biosciences). Hepatic levels of SAP130 were determined by ELISA (MyBioSource, San Diego, CA). Hepatic and serum nitrite levels were determined using the Griess assay (Life Technologies, Carlsbad, CA).
Greco S.H., Torres-Hernandez A., Kalabin A., Whiteman C., Rokosh R., Ravirala S., Ochi A., Gutierrez J., Salyana M.A., Mani V.R., Nagaraj S.V., Deutsch M., Seifert L., Daley D., Barilla R., Hundeyin M., Nikifrov Y., Tejada K., Gelb B.E., Katz S.C, & Miller G. (2016). Mincle Signaling Promotes Con-A Hepatitis. Journal of immunology (Baltimore, Md. : 1950), 197(7), 2816-2827.
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2

Measuring Nitrite Release in BV2 Cells

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Global cell activity was evaluated by determining nitrite release into the cell culture medium using the Griess assay according to the manufacturer's protocol (Life Technologies). Cells (1.8 × 104/well) were plated in 24-well culture plates and incubated overnight at 37°C. BV2 cells were incubated for 24 h in DMEM-High or -Low medium plus or minus lipopolysaccharide (LPS) (Sigma-Aldrich) (at either 0.1, 0.5 or 1 μg/mL; n = 6 experiments, treatments in triplicate). Culture medium was then collected, and 150 μL were transferred to 96-well microplates. Nitrite-containing medium was mixed with 20 μL of Griess reagent and 130 μL of deionized water. Standards (1-100 μM) were added to the microplate, which was next incubated for 30 min at RT. Nitrite concentration was determined by measuring A508 with a microtiter plate reader and analyzed with the SOFTmax Pro3.1.1 software.
Bordeleau M., ElAli A, & Rivest S. (2016). Severe chronic cerebral hypoperfusion induces microglial dysfunction leading to memory loss in APPswe/PS1 mice. Oncotarget, 7(11), 11864-11880.
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3

Assessing Macrophage Resistance to Pathogens

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Infection of murine macrophage-like RAW 264.7 cells was used to assess the resistance of the mutants to innate immune killing. Cells were grown and maintained in Gibco RPMI 1640 Glutamax (Thermo Fisher Scientific) with 10 % FBS (Thermo Fisher Scientific) at 37 °C and 5 % CO2. In total, 1×105 cells ml−1 were aliquoted into flat 96-well plates with the addition of 0.1 µg ml−1 recombinant murine IFNγ (RND Systems) and incubated overnight. Wild-type IP32956 and mutants were cultured overnight as previously described and inoculated at an m.o.i. of 0.1. RAW 264.7 cells with bacteria were incubated for 90 min to allow intracellular infection. Media and extracellular bacteria were removed and cells were washed three times with sterile PBS before fresh medium was added containing 100 µg imipenem ml−1 (Sigma). After 16 h, cells were lysed with 0.1 % Triton X100 in sterile, molecular-grade water for 10 min before manual disruption by pipetting and serial dilution of the resultant lysate for the c.f.u. assay. As a marker of cell activation state, we harvested cell culture supernatant at various time points and inferred nitric oxide production through the quantification of nitrite by a Griess Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Willcocks S., Huse K.K., Stabler R., Oyston P.C., Scott A., Atkins H.S, & Wren B.W. (2019). Genome-wide assessment of antimicrobial tolerance in Yersinia pseudotuberculosis under ciprofloxacin stress. Microbial Genomics, 5(11), e000304.
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4

Measuring Inflammatory Cytokine Levels

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HMC3 cells were plated into 6-well (2 × 105/well) dishes, grown for 20 h and treated with the tested compounds (1–10 μM) for 8 h followed by IFN-γ (100 ng/ml) stimulation. The release of NO in cell culture medium was evaluated by Griess assay according to manufacturer’s protocol (Thermo Fisher Scientific). In addition, the levels of IL-1β, TNF-α and IL-6 in medium pre-treated with 10 μM compound were determined using Human Instant ELISATM Kit following the manufacturer’s protocol (Invitrogen). The optical density at 450 nm was detected using Multiskan GO spectrophotometer.
Chiu Y.J., Lin S.A., Chen W.L., Lin T.H., Lin C.H., Yao C.F., Lin W., Wu Y.R., Chang K.H., Lee-Chen G.J, & Chen C.M. (2020). Pathomechanism characterization and potential therapeutics identification for SCA3 targeting neuroinflammation. Aging (Albany NY), 12(23), 23619-23646.
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5

Quantification of H2O2 and Nitrite

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H2O2 levels generated by He-O2 plasma treatment were measured using the Amplex Red assay following the manufacturer’s protocol (Thermofischer, Saint Aubin, France). Calibration curve was performed using hydrogen peroxide concentrations varying from 5 to 100 μM in PBS. Levels of nitrite NO2− were determined using the Griess assay according to the manufacturer’s protocol (Thermofischer, Saint Aubin, France).
Dezest M., Chavatte L., Bourdens M., Quinton D., Camus M., Garrigues L., Descargues P., Arbault S., Burlet-Schiltz O., Casteilla L., Clément F., Planat V, & Bulteau A.L. (2017). Mechanistic insights into the impact of Cold Atmospheric Pressure Plasma on human epithelial cell lines. Scientific Reports, 7, 41163.
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6

Quantifying Reactive Species in Plasma

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To quantify hydrogen peroxide (H2O2), one of the final products of the short-lived reactive species chemistry generated by the gas plasma, the amplex ultra red assay (ThermoFisher, Waltham, Massachusetts, USA), was used according to the manufacturer’s instructions. For the detection of two other stable molecules, nitrite (NO2-) and nitrate (NO3-), the Griess assay (ThermoFisher, Waltham, Massachusetts, USA) was performed according to the manufacturer’s instructions. As liquid carriers, PBS and cell culture media were used. Quantification was performed by comparing to standards with known H2O2, NO2- and NO3- concentrations. The ROS/RNS quantification assays were performed under equal CCP treatment regimes as for treatment of cells, i.e., 200 µL in 48-well plates.
Fischer M., Schoon J., Freund E., Miebach L., Weltmann K.D., Bekeschus S, & Wassilew G.I. (2022). Biocompatible Gas Plasma Treatment Affects Secretion Profiles but Not Osteogenic Differentiation in Patient-Derived Mesenchymal Stromal Cells. International Journal of Molecular Sciences, 23(4), 2038.
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7

Liver Glycogen and NO Measurement

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Liver was excised from each mouse, washed with ice-cold phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4; Sigma-Aldrich Co.), and weighted. One part of the liver was mashed using a 0.2 μm cell strainer and syringe rubber plunger in cold PBS, to obtain a liver homogenate. The liver homogenate was used to determine the levels of glycogen and NO by using ELISA kit (Sigma-Aldrich Co.) and Griess assay (Thermo Fisher Scientific), respectively.
Zulkawi N., Ng K.H., Zamberi N.R., Yeap S.K., Satharasinghe D.A., Tan S.W., Ho W.Y., Abd Rashid N.Y., Md Lazim M.I., Jamaluddin A., Alitheen N.B, & Long K. (2018). Antihyperglycemic and anti-inflammatory effects of fermented food paste in high-fat diet and streptozotocin-challenged mice. Drug Design, Development and Therapy, 12, 1373-1383.
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8

Quantification of Reactive Species

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Hydrogen peroxide levels generated by He-O2 plasma treatment were measured using the Amplex Red assay according to the manufacturer (Thermofischer, Saint Aubin, France). Calibration was performed in PBS using hydrogen peroxide concentrations varying from 5 to 100 μM. Levels of nitrite NO2- were determined using the Griess assay according to the manufacturer’s protocol (Thermofischer, Saint Aubin, France).
Dezest M., Bulteau A.L., Quinton D., Chavatte L., Le Bechec M., Cambus J.P., Arbault S., Nègre-Salvayre A., Clément F, & Cousty S. (2017). Oxidative modification and electrochemical inactivation of Escherichia coli upon cold atmospheric pressure plasma exposure. PLoS ONE, 12(3), e0173618.
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9

Measuring NO Production in HUVEC

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NO production in HUVEC was measured via Griess Assay (Thermofisher Scientific, G7921), DAF-FM Diacetate (Thermofisher Scientific, D23844), and a Zysense 280 NO analyzer. For DAF-FM, HUVEC were incubated with 1:1000 DAF-FM in EBM-2 medium for 30 minutes at 37°C. Cells were then fixed with 4% paraformaldehyde, mounted using Prolong Gold Antifade (Thermofisher Scientific, P36930), and imaged with a Zeiss LSM700 confocal microscope. For the NO analyzer, sample media was collected after flow exposure and centrifuged at 13,000 g for 5 minutes. 200 μL of supernatant was then added to the NO analyzer. Voltage readings were normalized to baseline, and the area under the peak was used to determine nitrite levels using a standard curve. Each sample was measured at least three times.
Basehore S.E., Bohlman S., Weber C., Swaminathan S., Zhang Y., Jang C., Arany Z, & Clyne A.M. (2021). Laminar Flow on Endothelial Cells Suppresses eNOS o-GlcNAcylation to Promote eNOS Activity. Circulation research, 129(11), 1054-1066.
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10

Profiling ROS and RNS in Plasma-Treated Liquids

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ROS and RNS were profiled in plasma-treated liquids (100 µL of PBS in a 96-well round bottom plate) by utilizing Amplex Ultra Red (Thermo Fisher Scientific, Waltham, NJ, USA) and the Griess assay (Thermo Fisher Scientific, Waltham, NJ, USA), as described previously [29 (link)]. The temperature of plasma-treated liquid was analyzed using an infrared thermometer to monitor the liquid temperature development.
Wolff C.M., Kolb J.F, & Bekeschus S. (2022). Combined In Vitro Toxicity and Immunogenicity of Cold Plasma and Pulsed Electric Fields. Biomedicines, 10(12), 3084.
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