Intraprep kit

For surface staining, cells were resuspended in flow cytometry (FACS) buffer (phosphate-buffered saline, pH 7.2, 0.2% bovine serum albumin, and 0.02% sodium azide) and incubated with FCR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature. After blocking incubation, surface markers were added for 15 minutes at 4 °C. Then cells were washed with the FACS buffer and were resuspended in each tube with 500 μl of FACS buffer. Intracellular staining for cleaved-caspase 3 was preceded by fixation and permeabilization with IntraPrep kit (Instrumentation Laboratory) and incubation for unconjugated primary antibody (rabbit IgG cleaved caspase-3 (Asp175) mAb, R&D Systems) 25 min at 4 °C. Cells were then washed and labeled with secondary antibody AlexaFluor 488 (Molecular Probes) for 25 min at 4 °C. Finally, cells were washed twice and resuspended in FACS buffer for acquisition. Data were obtained by using a FC500 (Beckman Coulter) flow cytometer and analyzed with Kaluza software. Three independent experiments were performed. The area of positivity was determined by using an isotype-matched mAb, and in total, 10⁴ events for each sample were acquired.

After stimulations, EC were permeabilized with IntraPrep kit (Instrumentation Laboratory) and incubated with unconjugated primary antibody p-ENOS (Abcam) for 25 min at 4 °C. Cells were then washed and labeled with secondary Antibody AlexaFluor 488 (Molecular Probes) for 25 min at 4 °C. Finally, cells were washed twice and resuspended in FACS buffer for acquisition. PBMCs were stained with the following monoclonal antibody, CD14 Monoclonal Antibody (61D3)-PE, (eBioscience™, Thermo Fisher Scientific, Italy), for 20 min in the dark at room temperature, washed twice, and resuspended in FACS buffer. Stained PBMCs were then acquired.
Data were obtained by using a FC500 (Beckman Coulter) flow cytometer and analyzed with Kaluza software. Three independent experiments were performed for both EC and PBMCs. The area of positivity was determined by using an isotype-matched mAb, and in total, 104 events for each sample were acquired.

After stimulations, pericytes were permeabilized with IntraPrep kit (Instrumentation Laboratory) and incubated with APC-conjugated anti-PDGFRβ (LSBio, Seattle, WA, USA) and FITC-conjugated anti-collagen I (Millipore, Millimarck, Germany) as previously described [21 (link)]. Data were analyzed with FC500 (Beckman Coulter, Brea, CA, USA) and Kaluza software. This assay was done in triplicate. The area of positivity was determined by using an isotype-matched mAb, and, in total, 10⁴ events for each sample were acquired. Data were obtained by using a FC500 (Beckman Coulter) flow cytometer and analyzed with Kaluza software. Three independent experiments were performed.