Ros assay kit

BMDMs were plated at 5 × 10⁴ per well in a black, clear-bottom 96-well plate and allowed to adhere overnight. After LPS stimulation, cells were incubated with 2',7'-dichlorodihydrofluorescein diacetate (H₂DFCDA) for 30 min before fluorescence measurement according to the manufacturer’s instructions (ROS Assay Kit, BioVision). Fluorescence values were corrected for background (unstained cells) and represented as percent of unstimulated flox control.

The reactive oxygen species (ROS) levels in the GCs were assayed according to the instructions of the ROS Assay Kit (BioVision, Milpitas, CA, USA). At the end of cell incubation in 96-well plates, 100 µL of the ROS assay buffer was slowly added after discarding the medium for 5 min. The supernatant was then discarded, and the cells were rinsed slowly once with PBS. Subsequently, 100 μL of 1 × ROS labeling buffer was added, and the cells were left to incubate at 37 °C for 45 min, avoiding light. The supernatant was discarded again, 100 μL of ROS inducer buffer was added, and the mixture was transferred at 37 °C for 1 h under light protection. The cells were then observed under an inverted fluorescence microscope.

The intracellular ROS generation was detected using a ROS assay kit (Abcam) according to the manufacturer's instructions. Briefly, A549 and H1299 cells were seeded into 6-well cell culture plates with 2 × 10⁵ cells per well and cultured continuously for 12 h. Different intervention reagents were added to the cultural plates and cultured continuously for 48 h. Tumor single cell suspensions were incubated with DCFH‐DA probe at 1:1000 dilution in serum‐free RPMI medium at 37°C for 30 min, then washed with serum‐free RPMI medium three times and detected by a fluorescent reader (TECAN) with excitation at 488 nm.

Reactive oxidative stress (ROS) Assay Kit (ab186027, Abcam, USA) and Lipid Peroxidation (MDA) Assay Kit (ab118970, Abcam, USA) were used to evaluate the oxidative stress levels as per the manufacturer’s instruction. For ROS assay, cultured cells were stained with ROS Red working solution and fluorescence intensity was measured at 520/605 nm (Ex/Em). For lipid peroxidation assay, 600 μL of thiobarbituric acid solution was added to 200 μL samples or standards. After incubation at 95 °C for 1 h, samples and standards were cooled to room temperature in ice bath. 200 μL of the reaction mixture from each sample or standard was moved into a 96-well plate and optical density (OD) value was immediately measured at 532 nm using a plate reader (Thermo Fisher Scientific, USA).

The relative iron concentration in macrophages was measured with the Iron Assay Kit (ab83366, Abcam). Briefly, cells were rapidly homogenized in an iron assay buffer and centrifuged at 16,000×g for 10 min at 4 ℃. The supernatant was collected and then iron assay buffer was added respectively to measure ferrous iron. Iron probes were added and incubated at 37 ℃ for 1 h. Optical density was measured at 593 nm, and a standard curve line was used for the iron concentration calculation.
The level of intracellular ROS was determined using a ROS assay kit (Abcam) according to the manufacturer's instructions. In brief, cells were washed and incubated with 2,7-dichlorofluorescein diacetate (DCFDA, Thermo Fisher Scientific, USA) at 37 °C for 30 min. The production of ROS was measured using flow cytometry.

Skeletal muscles were homogenized, and ROS level analysis was performed using 2′,7′-dichlorofluorescein diacetate fluorescent dye as a probe and fluorescence density was measured at 490/520 nm according to manufactory guidance (ROS assay kit; Abcam, Cambridge, MN, USA) and the measured values of optical density (a relative fluorescence unit (RFU)) were corrected by the protein concentrations of samples and were expressed as RFU/μg protein [44 (link)].

After culturing cells in a 96-well plate, the levels of reactive oxygen species (ROS) were measured using a ROS assay kit (Abcam) in accordance with the manufacturer's instructions.