Ros detection cell based assay kit

Quantification of lactate level was done using Lactate Colorimetric Assay Kit (Biovision, Inc., Milpitas, CA, USA) according to vendor’s protocol. Adherent or suspended cells (2x10⁵ cells) were incubated in the culture medium at pH7.0 or pH8.5 for 1 day without pH adjustment by CO₂. The conditioned medium was collected and centrifuged for the following assay.
Detection of ROS levels in melanoma cells was done using ROS Detection Cell-Based Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to vender’s protocol. Dihydroethidium is the redox-sensitive fluorescent probe. Its oxidation into 2-hydroxyethidium by superoxide or other ROS causes the increased fluorescence intensity at 600nM or 576nm. Antimycin A treatment and N-acetylcysteine treatment were positive and negative controls of ROS production, and used to calculate the relative percentage of ROS production.

ROS levels were measured using chloromethyldihydrodichlorofluorescein diacetate (CM‐H2DCF/DA) assay by ROS Detection Cell‐Based Assay Kit (Item# 601520, CAYMAN CHEMICAL) according to the manufacturer’s protocol.

Cancer cell lines (2 × 10⁴) were plated on collagen type I coated 96 well plate (IWAKI) and incubated with DMEM containing 5% FBS for a day. The medium was replaced with DMEM 5% FBS containing compounds or 500 µM H₂O₂ as a positive control. After treatment of compounds and UV irradiation, the cells were treated with the ROS detection cell-based assay kit (Cayman chemical) according to the manufacturer’s instructions. Negative control was maintained by N-Acetyl cysteine to one group of untreated or treated cells. The cells were incubated for 1 h at 37 °C in dark, the ROS staining buffer was aspirated and 100 μl of DMEM 5% FBS was added to all the wells, the fluorescence was measured at 575 nm (Excitation: 488 nm). The ROS generated was expressed as total DHE fluorescence exhibited by the control and treated cells.

ATP levels in HCAECs were measured with the ATP Assay Kit (S0026; Beyotime Biotechnology, Haimen, Jiangsu, China) according to the manufacturer's instructions. Total ROS levels in HCAECs were measured using the ROS Detection Cell-Based Assay Kit (601290; Cayman Chem., Ann Harbor, MI, USA) according to the manufacturer's recommendations.

ROS generation in astrocytes was detected by using the ROS Detection Cell-Based Assay kit (Cayman, USA) according to the manufacturer’s instructions. For nuclear counterstaining, the cells were stained with DAPI. Finally, the samples were observed and photographed under a fluorescence microscope.

The ROS levels in brain hemispheres were determined using 2,7 –dichlorofluorescein diacetate (DCF-DA), which is a fluorogenic dye that upon coming into contact with ROS is converted into highly fluorescent 2’, 7’–dichlorofluorescin (DCF) detected by fluorescence spectroscopy (ROS Detection Cell-Based Assay Kit, Cayman Chemical, USA) [27 (link)]. DHE has been shown to be oxidized by superoxide or through non-specific oxidation by other sources of reactive oxygen species.
Brains were collected 3 h after the injection of the mGluR2/3 agonists and tissues from the left and right hemispheres were homogenized separately in ice-cold 40 mM Tris-HCL buffer (pH.7.4). The resulting brain homogenates were incubated with DCF-DA (25 μM) for 30 min at 37°C. The formation of the fluorescent product DCF was monitored using a fluorescence spectrometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The relative fluorescence unit (RFU) was calculated per 1 mg of protein in the homogenate. The protein concentration was determined according to Bradford [28 (link)]. The effect of H-I and used agonists was estimated through the determination of the ROS level in individual brains and then standardized for variation at the basal level measured in brains from sham operated animals.