FACS analysis

Measurement of intracellular H₂O₂ levels in 5 x10⁴ ECFC-ECs in suspension at passage 4 was performed after loading with 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA, 20 μM; MitoSciences, Cambridge, MA, USA) for 30 min at 37°C in the dark, washed with PBS, and detached with TrypLE Express. After centrifugation, cell pellets were suspended in 200 μl PBS. Formation of ROS was detected by the signal obtained from the fluorescent reaction products 2’,7’-dichlorofluorescin (DCF) and ethidium. Cells were washed and analyzed by means of fluorescence-activated cell sorting (FACS) (FACS Calibur, Becton Dickinson, USA). Results were analyzed using the FlowJo Software v.7.6.5 (Ashland, OR, USA). The absolute values of mean fluorescence intensity (MFI) were expressed as percentages and the result obtained from controls was considered as the 100% reference value. For determination of apoptosis, ECFC-EC in passage 4 were detached with TrypLE Express for 5 min at 37°C, washed in PBS, and suspended in binding buffer (BD Biosciences). After centrifugation, cell pellet was suspended in 50 μl binding buffer and incubated for 15 min at room temperature with 2 μl annexin–fluorescein isothiocyanate (FITC; BD Biosciences) and 2 μl propidium iodide (PI; BD Biosciences). Binding buffer (300 μl) was added and cells were washed twice and analyzed by means of FACS. We analyzed the results using FlowJo Software v.7.6.5.

To evaluate the expression of γH2AX in ECFC-ECs from controls and VTD patients at passage 4, cells were grown with type I rat tail collagen in 6-well plates at 80% of confluence. Cells were rinsed twice in PBS, fixed in 4% paraformaldehyde in PBS for 20 min, and permeabilized with 0.1% Triton 100 X (Sigma Aldrich USA), in PBS for 20 min at 4°C. Then, cells were blocked in a 1% BSA solution in PBS and incubated with a murine anti-human anti-H2AX (pS139) PE-conjugated antibody (BD Biosciences). Labeled cells were analyzed in a FACSVerse (BD Biosciences), and the data obtained were evaluated with a Flowjo software (Version 10.3).