Ros glo h2o2 assay kit

MDA-MB-231 cells were transferred to 96-well plates (20,000 cells/well) after treatment, with 60 μL fresh-media and were incubated for 12 h, as described previously^{20 (link),55 (link)}. At 7 h, 20 μL of H₂O₂ substrate solution from ROS-Glo H₂O₂ assay kit (Promega) was added to cells and incubated for 5 h more. At 12 h, cells were incubated for 20 min after addition of ROS-Glo™ detection solution and the Lum was measured at 0.5 s integration. Synergy LX Multi-Mode Reader was used for this purpose.

For the detection of H₂O₂ production, the ROS-Glo H₂O₂ assay kit (Promega, Madison, WI, USA) was used according to manufacturer’s recommendation with the indicated changes. Briefly, 15,000 cells/well were seeded in 96-well white-walled plates with clear bottoms and cells were allowed to adhere overnight. The following day, cells were treated with indicated concentrations (µM) of KP372-1 or KP372-1 + DIC or KP372-1 + N-acetylcysteine amide [NAC, 1 mM or 5 mM for total of 5 h (pre-treatment for 3 h and co-treatment for 2 h)] or DMSO (as control) for specified time (min) points in a total volume of 50 µl that contained 10 µl of H₂O₂ substrate. Then, 50 µl of ROS-Glo detection solution was added to each well and cells were incubated for 20 min at room temperature. Luminescence was measured using a Victor X5 plate reader. Luminescence values of treated samples were normalized to luminescence values of control samples to generate reported graphs. The reported values are the results of n = 4.

Intracellular ROS levels were measured using ROS-Glo™ H₂O₂ Assay Kit (Promega, Madison, WI, USA). Intracellular ATP levels were detected using CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega). The alterations in intracellular GSH/GSSG levels were examined using GSH/GSSG-Glo™ Assay Kit (Promega). All experiments were performed according to the manufacturer’s instruction. The luminescent intensity was measured via a microplate reader (BioTek) at the indicated time points.

Cell lines HEK293T, IL16lamp2b and EXO-Tat were cultured in Dulbecco’s Modified Eagle medium (GE Healthcare Bio-Sciences, PA, USA) with 10,000 U/mL penicillin-streptomycin (Thermo-Fisher, USA), 200 mM of L-Glutamine (Thermo-Fisher, USA), and fetal bovine serum over a period of 48 hours. Each of the cell lines were cultured in five plates and considered as five replicates. After dissociation with 0.25% trypsin (Thermo-Fisher, USA), cells were centrifuged and re-suspended in PBS buffer (GE Healthcare Bio-Sciences, PA, USA). An equal number of cell (3.0 × 105/mL) concentrations were used for each assay.
Cell Viability assay was preformed using the CellTiter-Glo 2.0^® bioluminescence assay kit (Promega, WI, USA) according to the manufacturer’s protocol. Readings were taken using the Glo-Max Explorer system (Promega, WI, USA), wherein higher luminescence correlated to viability [31 (link)]. Readings were taken using the Glo-Max with higher luminescence correlating to more cell death [32 (link)]. Reactive Oxygen Species (ROS) were measured using ROS-Glo™ H₂O₂ assay kit (Promega, WI, USA) according to the manufacturer’s protocol wherein higher signals are indicative of increased amount of ROS in cells [33 (link)].

H₂O₂ was evaluated using ROS-Glo™ H₂O₂ Assay Kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol using a microplate reader. Sertoli cells plated at 1×10⁵ cells cells/well to a 96-well white plate and exposed to various concentrations of OLA (100-, 200-, 400-, 800 μg/ml) for 18 h before termination, 10 mM of NAC was added 1 h before OLA treatment. Less than 80μl of medium is desirable to accommodate addition of test compounds. Add 20 μl of H₂O₂ Substrate solution to cells and mix. The final well volume will be 100 μl, and the final H₂O₂ Substrate concentration will be 25 μM, incubate at 37°C in 5% CO₂ for the final 6 h of treatment. Add 100 μl of ROS-Glo™ Detection Solution to each well and incubate for 20 mins at room temperature, and the luminescence was determined with EnSpire ® Multimode Reader (PerkinElmer, Inc., Waltham, USA).

LPS, MTT, penicillin/streptomycin, trypsin-EDTA, hematoxylin, eosin, and protease inhibitor were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM, FBS, and other cell culture reagents were supplied by Gibco (Carlsbad, CA, USA). DMSO was obtained from Bioshop (Burlington, Ontario, Canada). RNA extraction kit (RiboEx and Hybrid-R) was bought from Gene All (Seoul, Korea). Griess reagent, cDNA synthesis kit (ReverTra Ace qPCR RT Kit), T-PER, and BCA protein assay kit were purchased from Thermo Scientific (Waltham, MA, USA). SYBR Green qPCR Kit obtained from TOYOBO (Tokyo, Japan). Primary antibodies ERK1/2, JNK, p38, COX-2, IκBα, NF-κB, and β-actin were supplied by Cell Signaling (Danvers, MA, USA). Secondary antibody (goat anti-rabbit immunoglobulin g horseradish peroxidase) was provided by Santa Cruz (Dallas, TX, USA). WESTSAVE gold ECL detection kit was obtained from Abfrontire (Seoul, Korea). TBARS assay kit by Cayman (Ann Arbor, MI, USA). ROS-Glo H₂O₂ assay kit was supplied by Promega (Madison, WI, USA). Zoletil 50 was bought from Virbac (Carros, France).