Annexin 5 cy5

Effects of the HF blood plasma (1 h and 24 h exposure) on the endothelial cell viability was assessed by annexin/PI apoptotic assay. Five samples were randomly selected from each blood plasma group and used in this assay. Ea.hy926 cells were seeded onto 6-well plates and grown for 24 h at 37°C. Then the cells were serum-starved for 2 h, followed by blood plasma addition to the medium (1% final concentration). After 1 h incubation with the blood plasma, cells were harvested and washed three times with ice-cold 1x PBS and resuspended in 1x annexin binding buffer (Biovision, Mountain View, CA). 1x10⁵ of cells were collected and incubated with 5 μL of Annexin V-Cy5 (Biovision) and 1 μL (100 μg/mL) of propidium iodide (PI, Invitrogen^TM) in the dark for 15 min at room temperature, after which 1x annexin binding buffer was added to make a total volume to be 300 μL. The percentages of apoptotic and necrotic cells were measured by flow cytometry (BD FACS CantoTM, Becton Dickinson, Franklin Lakes, NJ). The same protocol was applied to the cells, which were incubated with experimental blood plasma for 24 h. The experiments were performed in triplicates.

Cells were stained with annexin V-Cy5 (BioVision, 1013; 1:1000 dilution) and propidium iodide (Immuno Chemistry Technologies, 638; 1:500 dilution) in annexin V binding buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂ for 15 min on ice. To assess mitochondrial membrane potential, cells were stained with 100 nM tetramethylrhodamine methlester (TMRM; Thermo Fisher Scientific, T668) in culture medium for 15 min room temperature. Bone marrow-derived mast cells suspended in staining buffer (PBS, 1% BSA, 0.1% NaN₃) were pretreated with TruStain fcX (BioLegend, 101320) at 4 °C for 30 min and then stained with APC anti-mouse CD117 (BioLegend, 105811) and FITC anti-mouse FcεRIα (BioLegend, 134305) at 4 °C for 30 min. The cells were analyzed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences), and data were analyzed using FlowJo V10 software.