Monocytes (2.5 x 105cells/mL) were suspended in Hank’s balanced salt solution (HBSS) without Phenol Red (Gibco, ThermoFisher, Waltham, USA0. To detect the intracellular ROS production, cells were loaded with a 5 µM CellROX probe (ThermoFisher, Waltham, USA) for 30 minutes prior to treatment. The medium with non-internalized probe was removed and the cells were pretreated with or without TAK-242 (1 µM; Cayman Chemical Company, Ann Arbor, MI, USA) and then incubated with RBC 0-EVs or RBC 21-EVs for 15 minutes at 37°C in a 5% CO2 atmosphere During treatment, probe oxidation was monitored using a Flexstation™ Multi-label Plate Reader (ThermoFisher, Waltham, USA) at excitation and emission wavelengths of 640 and 665 nm, respectively.
Cellrox probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CellROX probe is a fluorogenic dye used to measure oxidative stress in cells. It becomes fluorescent upon oxidation, providing a quantitative assessment of reactive oxygen species levels within the cellular environment.
Automatically generated - may contain errors
2 protocols using cellrox probe
1
Monocyte Intracellular ROS Detection
2
Quantifying Cellular and Mitochondrial ROS
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ROS activity in cells and mitochondria was measured as previously described (24 (link)). In brief, grouped cells were loaded with 5 µmol/mL of Cell Rox probe (C10444, Thermo Fisher Scientific) or Mito Sox probe (M36008, Thermo Fisher Scientific) for 30 min or 10 min at 37 ℃ in the dark, after which the probes were discarded. Next, the cells fixed by paraformaldehyde (4%, 15 min) were stained by DAPI (1 µg/mL) for 15 min in the dark and then treated with an anti-fluorescence quencher, followed by fluorescence microscope (Olympus Corporation, Japan) observation. The average fluorescence intensity was analyzed by ImageJ software.
To determine the ROS activity in myocardial tissues, the following steps were performed using a ROS kit (mlbio). Specifically, the myocardial tissues were mixed in a certain amount of PBS solution (pH =7.4), followed by 20-min centrifugation (2,000–3,000 rpm/min). The supernatant was gathered and diluted at a ratio of 1:1. Subsequently, 50 µL samples were added to reaction wells and cultured for 1-h at 37 ℃. Afterwards, the plates were washed for 3 times with 30 s for each time. After color development, 50 µL of stop buffer was dropped immediately, and the OD value of each well was determined at 450 nm wavelength.
To determine the ROS activity in myocardial tissues, the following steps were performed using a ROS kit (mlbio). Specifically, the myocardial tissues were mixed in a certain amount of PBS solution (pH =7.4), followed by 20-min centrifugation (2,000–3,000 rpm/min). The supernatant was gathered and diluted at a ratio of 1:1. Subsequently, 50 µL samples were added to reaction wells and cultured for 1-h at 37 ℃. Afterwards, the plates were washed for 3 times with 30 s for each time. After color development, 50 µL of stop buffer was dropped immediately, and the OD value of each well was determined at 450 nm wavelength.
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