Total ROS Assay kit 520 nm (No. 88-5930-74) was purchased from Thermo Fisher, USA. Biochemical assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Apoptosis Kit (No. 556547) was purchased from Becton Dickinson Company (Franklin Lakes, USA). Antibodies against Nod-like receptor protein 3(NLRP3) and nuclear factor-κB (NF-κB) were obtained from Sagon Biotech (Shanghai, China). The antibody against Gasdermin D was obtained from Thermo Fisher (USA) and β-actin from Bioss (Beijing, China). Caspase-1 p20 antibody was purchased from Boster Biological Technology Co., Ltd (Wuhan, Hubei, China). Ageratina adenophora was collected from Xichang, Sichuan Province, Southwest China. The collected leaves were cleaned, ground and screened at room temperature to make dry powder. The powder was stored in shade condition with an ambient temperature of 20 ± 2°C. For the preparation of 10%, 20% and 30% diet pellet, A. adenophora and mice feed were homogenized in water solution by the ratio of 1 : 9, 1 : 8 and 1 : 7, respectively. Then the diet was cast in the form of cylinders and dried at 27°C for 48 h.
Total ros assay kit 520 nm
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Total ROS Assay kit 520 nm is a laboratory product designed to measure total reactive oxygen species (ROS) levels in biological samples. It utilizes a fluorescent probe that reacts with a wide range of ROS, generating a quantifiable fluorescent signal at 520 nm. This kit provides a standardized method for researchers to assess overall oxidative stress in their samples.
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Lab products found in correlation
Biochemical assay kit, by Nanjing Jiancheng (1 mentions)
Apoptosis kit, by BD (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Edta tube, by BD (1 mentions)
Near ir dead cell stain, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 2.4g2, by BD (1 mentions)
Lsrii flow cytometer, by Tree Star (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apoptosis detection kit efluor 450, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Phosflow perm buffer 3, by BD (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
No detection kit, by Nanjing Jiancheng (1 mentions)
Software, by FlowJo (1 mentions)
7 protocols using total ros assay kit 520 nm
1
Ageratina adenophora ROS Assay
2
Apoptosis and ROS Detection
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Apoptosis was detected with a PE‐Annexin V apoptosis detection kit (BD Pharmingen), and reactive oxygen species (ROS) were measured with the Total ROS Assay Kit 520 nm (Thermo Fisher), according to the manufacturer's instructions, followed by FACS analysis using a flow cytometer (ACEA Bioscience).
3
Evaluating Bacterial ROS Generation
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Production of reactive oxygen species (ROS) by treated and untreated (control) bacteria was evaluated using fluorescent dye-based assay (total ROS Assay Kit 520 nm; Thermo Fisher Scientific, Carlsbad, CA, United States), according to the manufacturer’s recommendations. Briefly, 2 ml bacterial cells at approximately 109 CFU/ml were pelleted (5,500 × g, 6 min, 25°C) and resuspended with an ROS stain solution at a final concentration of 1×. Thereafter, the cells were incubated to be labeled (37°C, 1 h, in absence of light). After label time, extracellular fluorescent dye was removed with two washing steps by centrifugation (5,500 × g, 6 min, 25°C) and PBS. Finally, the labeled cells were resuspended in 1 ml of PBS. A volume of 0.1 ml of the labeled cells was added to 0.1 ml of PBS containing the antimicrobials (treatments) or PBS alone (untreated control). At nine time points of treatment (0, 15, 30, 45, 60, 75, 90, 105, and 120 min), fluorescence emission was measured at 520 nm using a fluorescent microplate reader (PERKIN ELMER 1420 MULTILABEL COUNTER VICTOR 3) with an excitation wavelength of 495 nm. The experiment was conducted in triplicate, at least on three different occasions.
4
Quantifying Cellular Oxidative Stress
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Peripheral blood samples were obtained by venipuncture and collected into EDTA tubes (BD; cat# 366450). A 50 µL aliquot of whole blood was stimulated with an equal volume of ROS Assay Mix containing a 1:250 dilution of ROS Assay Stain into ROS Assay Buffer (Total ROS Assay Kit 520 nm; Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA; cat# 88-5930-74). Incubation proceeded at 37 °C with 5% CO2 for 60 min before erythrocytes were lysed by the addition of Ammonium–Chloride–Potassium (ACK) lysing buffer (Fisher Scientific; cat# A1049201). Following erythrocyte lysis, the remaining leukocytes were centrifuged at 300×g for 5 min and resuspended in 0.5 mL of 1X PBS. Finally, ROS production by monocytes and neutrophils was detected using a BD LSRFortessa flow cytometer and FACSDiva software version 7.0. FlowJo software version 10 was used to perform data analysis and to create figures.
5
Multiparametric Immune Cell Analysis
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Cells were incubated with 0.5 µg/ml anti‐CD16/32 (2.4G2, BD Bioscience), Near IR Dead cell stain (Invitrogen) and fluorescently labeled antibodies (see Supporting Information: Table S4 ). For cytokine analysis, cells were cultured for 4 h with 10 μM Brefeldin A before staining. Cells were fixed and permeabilized with Foxp3/Transcription Factor Buffer Staining Set (eBioscience). For apoptosis the Annexin V Apoptosis Detection Kit eFluor 450 (Invitrogen) was used. ROS was assayed using Total ROS Assay Kit 520 nm (Invitrogen). For pDok cells were fixed with BD PhosFlow Lyse/Fix buffer then Foxp3/Transcription Factor Buffer Staining Set (eBioscience) before a permeabilization with BD PhosFlow Perm Buffer III and staining with pDok1 Y398, then anti‐rabbit AF488. Lymph node cells were stained rather than skin to avoid potential effects of lengthy enzymatic digestion of skin on pDok1 levels. Cells were analyzed using a BD Fortessa or LSRII flow cytometer and FlowJo (TreeStar).
6
Quantification of Inflammatory Markers
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All samples were frozen at −80 °C until all biological replicates have been acquired. Then, detection for Cytokines, ROS and NO were performed immediately. The concentration of TNF‐α (R&D Systems, MN), MMP9 (Boster, Wuhan, China), and IL‐1β (R&D Systems, MN) were detected using an ELISA as per manufacturer's instructions. Absorbance was read at 490 nm on a spectrophotometer, and sample concentrations were calculated using an equation generated from a standard curve. ROS was determined by FACS using the total ROS Assay Kit 520 nm (Affymetrix, 88‐5930) as per manufacturer's instruction. NO was measured by nitrate reductase method using NO detection kit (Jiancheng Bioengineering, Nanjing, China). Briefly, the absorbance was read at 550 nm on a spectrophotometer, and the content of NO2− is determined colorimetrically.[90 ]
7
ROS Production Analysis by Flow Cytometry
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ROS production was analysed by flow cytometry. Briefly, cells (106 cells/sample) were incubated with DMSO or ROS assay stain solution for 1 h using the Total ROS assay kit 520 nm (affymetrIX eBioscience, San Diego, USA) following the manufacturer's instructions. Cells were harvested, centrifuged for 5 min at 300 relative centrifugal force, resuspended in PBS with ROS assay stain solution and incubated for 1 h at 37°C. Cell staining was assessed by flow cytometry. Results were analysed using the FlowJo software (FlowJo LLC, Ashland, OR, USA).
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