Apc conjugated anti α sma

Twenty-four hours after treatment, HUVECs were harvested and stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (1:100, Biolegend, Cat#:343504, San Diego, CA, USA), APC-conjugated anti-α-SMA (1:1000, Abcam, Cat#: ab202296), and FITC-conjugated anti-Ki67 (1:100, Invitrogen, Cat#: 11569882, Waltham, MA, USA). Cells were analyzed by BD FACSAria II flow cytometer (BD Biosciences, Franklin Lake, NJ, USA), viable singlet cells were selected by FSC/SSC gating, and results were further analyzed by FlowJo 10 software.

Raw264.7 macrophages and bone marrow differentiated monocyte/macrophages were maintained in culture media with 10% heat-inactivated FBS at 37°C in an incubator with a humidified atmosphere and 5% CO₂. 10^–7 M aldosterone (Ald) was used for Ald treatment and in Ald + Spir group cells were pre-treated with 10 uM spirolactone 1 h prior to Ald treatment. 24 h after the treatment, all cells were harvested and stained with PE-conjugated anti-moue F4/80 (Biolegend), APC-conjugated anti-α-SMA (Abcam) for 1 h in dark tubes. Cells were analyzed in a BD Accuri C6 flow cytometer, viable singlet cells were selected by FSC/SSC gating, and data were further analyzed using FlowJo 10 software.

Twenty-four hours after treatment, RAW264.7 cells and BMDMs were harvested and stained with fluorescein isothiocyanate (FITC)-conjugated anti-F4/80 (1:100, Invitrogen, Cat#: 11-4801-82), APC-conjugated anti-α-SMA (1:1,000, Abcam, Cat#: ab202296) and anti-CD206 (1:100, Abcam, Cat#: ab64693) or iNOS (1:100, Abcam, Cat#: ab15323) for 1 h, and then the secondary antibody goat anti-rabbit IgG H&L (PE) was preabsorbed (1:500, Abcam, Cat#: ab72465) for 1 h in the dark. Unstained cells were used as negative controls. Cells were analyzed on a BD FACSAria II flow cytometer (BD Biosciences, Franklin Lake, NJ, USA), viable singlet cells were selected by FSC/SSC gating, and data were further analyzed by FlowJo 10 software.