Dcfh da probe

After relevant treatment, LDH activity of supernatant was measured by LDH kits (Jiancheng). Cardiomyocytes were detected as follows: cell viability was tested by MTS kit (Promega), caspase-3 activity was detected by caspase-3 activity kit (R&D), apoptosis was measured with Annexin V-EGFP/PI apoptotic detection kit (BD Biosciences, San Diego, CA, USA), and intracellular ROS was assessed using a DCFH-DA probe (Invitrogen, Carlsbad, CA, USA), according to their manufacturer's instructions, respectively [15 (link)].

Cells stably transduced with wild-type or mutated mitoStat3 and non-transduced cells were seeded in quadruplicate in 60 × 15 mm dishes (20 × 10³ cells/cm²) in complete culture medium. Twenty-four hours after seeding, half dishes were irradiated with UVC (10–20 J/m²), then irradiated and non-irradiated dishes were processed for ROS generation by using the Dichlorodihydrofluorescein diacetate (DCFH-DA) probe (Invitrogen, Life Technologies).

Intracellular stress was assessed by measuring reactive oxygen species (ROS) levels following AgNP exposure under the denoted conditions. A549 and U937 cells were seeded in 24-well plates at 2 × 10⁵ cells per well or 1.5 × 10⁵ cells/mL, respectively, and returned to the incubator until the following day. Next, the cells were washed and incubated with the DCFH-DA probe (Thermo Fisher Scientific) for 30 min, washed again, then dosed with the AgNPs at 0, 5, or 25 µg/mL within either a static or dynamic environment. After 24 h incubation, the ROS levels were measured via fluorescent analysis using a Synergy 4 BioTek microplate reader. Untreated cells under static conditions served as the negative control for normalization and hydrogen peroxide-dosed cells served as a positive control.

Stress levels within the HepG2 cells were assessed via two endpoints: reactive oxygen species (ROS) and actin levels. For both metrics HepG2 cells were seeded into black 96-well plates at a density of 3x10⁴ cells per well and returned to the incubator overnight. For ROS analysis, the cells were washed and incubated with DCFH-DA probe (Thermo Fisher Scientific), washed again, and exposed to the stated PtNP conditions for 24 hours.
For actin evaluation, the HepG2s were exposed to the denoted conditions for 24 hours, washed, and fixed with 4% paraformaldehyde. The cells were then probed for actin using Alexa Fluor 555-phalloidin (Thermo Fisher Scientific), in accordance with the manufacturer recommendations. After proper treatment and staining, both ROS and actin levels were quantified via fluorescence analysis on a Synergy 4 BioTek microplate reader. PtNP dosed conditions were normalized against an untreated, negative control.

C28/I2 chondrocytes were plated in a 96 well plate as described above. DCFH-DA probe (#D399, Thermo Fisher Scientific, USA) was employed for the determination of intracellular ROS production [15 (link)]. After incubation with 10 μM DCFH-DA for 30 min, C28/I2 chondrocytes were observed with an inverted fluorescence microscope (Olympus, Tokyo, Japan). The levels of ROS were calculated using the software Image J. Firstly, we defined the regions of interest in the fluorescent images. The integrated density value (IDV) of fluorescence was calculated. Total cells (n) in the regions were then counted. Average ROS = IDV/n.

Intracellular stress was determined via reactive oxygen species (ROS) levels following AgNP exposure and for basal stress assessment. The cellular systems were seeded in 24-well plates at 2 × 10⁵ cells per well and incubated overnight. The next day, the cells were washed and incubated with the DCFH-DA probe (Thermo Fisher Scientific) for 30 min. After staining, the cells were again washed, dosed with the AgNPs in the denoted exposure environments, and incubated for 24 h. At the end of the exposure duration, ROS levels were measured via fluorescent analysis using a Synergy 4 BioTek microplate reader. Untreated, static A549 cells served as the negative control for normalization.

Levels of intracellular and mitochondrial ROS were measured using a DCFH-DA or mitoSOX probe as described in the previous method [43 (link)]. In brief, at 4, 8, 16, 24, and 48 hours after corresponding treatment, cells were harvested, collected, and washed with serum-free DMEM. Then, cells were mixed with serum-free media containing 10 μM DCFH-DA probe (Molecular Probes, Eugene, OR, USA) or 5 μM mitoSOX probe (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37°C in the dark for 30 min with slight agitation every 5 min. Subsequently, cell pellets were collected, washed three times with PBS, and resuspended in 500 μl PBS for flow cytometry analysis (Cytomics FC500). The induced green fluorescence from 10,000 cells was documented at 488 or 510 nm. FlowJ software was used to analyze the average fluorescence intensity.