Invitrogen attune nxt software

Mitochondrial ROS (mROS) generation was assessed using MitoSOX Red mitochondrial superoxide indicator (#M36008, Invitrogen). Control and Ang II (±Ang 1-7)-treated thoracic VSMCs were trypsinized, and monolayers, as well as floating cells in the cell culture medium, were centrifuged at 1500× g for 5 min. Cell pellets were washed and resuspended in HBSS/Ca²⁺ buffer at 0.5 × 10⁶ cells/mL. Cells were then incubated with 1 µM MitoSOX Red at 37 °C for 10 min in the dark. After the incubation, cells were washed and resuspended in HBSS/Ca²⁺ buffer for flow cytometric analysis using the Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA, USA) at excitation and emission wavelengths of 510/580 nm. The Invitrogen™ Attune™ NxT Software (version 2.6, ThermoFisher Scientific, Waltham, MA, USA) was used to analyze the MitoSOX fluorescence intensity, representing the mitochondrial ROS levels.

Cells were harvested post-treatment, fixed in ice-cold 70% (v/v) ethanol and stored at −20 °C. Prior to analysis, cells were washed with phosphate buffered saline (PBS), resuspended in 500 μL PBS with 50 μg/mL propidium iodide (Sigma-Aldrich) and 50 μg/mL RNAse A (Sigma-Aldrich), and incubated at 37 °C for 30 min. Samples were analysed on the Attune NxT Flow Cytometer using Invitrogen™ Attune NxT Software (Thermo Fisher Scientific). Data were analysed using FlowJo ^TM (BD Biosciences, Wokingham, UK). Experiments were at least n = 3.

Thoracic aortic SMC proliferation was evaluated using the Ki67 staining and flow cytometric analysis. Briefly, VSMCs were plated at 25,000 cells per well in 6 well plates and treated with saline and Ang II (±Ang 1-7) for 24 h. Control and Ang II (±Ang 1-7)-treated cells were trypsinized, and monolayer cells in the cell culture medium were centrifuged at 1500g rpm for 5 min. The cell pellets were fixed with 70% ethanol and incubated at −20 °C for 2 h. After fixation, cells were washed twice with staining buffer (1% FBS, 0.09% NaN₃ in PBS) and resuspended in staining buffer at 1 × 10⁶ cells/mL. The cells were then incubated with the Anti-Ki67 antibody (#ab16667, Abcam, Trumpington, CB, UK) at room temperature for 30 min. After the incubation, cells were washed twice with staining buffer. Alexa Fluor 488-conjugated Goat anti-Rabbit secondary antibody (#A11034, Invitrogen, Waltham, MA, USA) was added, and cells were incubated at room temperature for 30 min in the dark. After incubation, cells were washed twice with staining buffer, and samples were analyzed within 1 h using an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA, USA) at 488 nm excitation and 530 nm emission wavelength. Quantitative analysis of flow cytometric data was performed using Invitrogen™ Attune™ NxT Software (version 2.6, ThermoFisher Scientific, Waltham, MA, USA).