Zen software v 3.3 blue edition

To study HL-60 adhesion to endothelial cells, HULEC cells were seeded in 24-well cell imaging plates (Merck Millipore, Darmstadt, Germany) and treated as described above. After washing with PBS, carboxyfluorescin succinimidyl ester (CFSE)-labeled HL-60 cells (4.5 × 10⁴) were added at 300 µL of FBS-free medium. Cells were co-incubated for 30 min at 37 °C, 5% CO₂ and then nonadhered HL-60 cells were removed and plates were rinsed twice with 1 mL FBS-free medium. Then, HL-60 cell adhesion to endothelial monolayers was measured as an area of residual green fluorescence staining using an Axio Observer 7 fluorescence microscope and ZEN software v.3.3 blue edition (Carl Zeiss Inc., Dresden, Germany).

Immunofluorescent staining of endothelial total and phosphorylated at Ser1177 nitric oxide synthase (total-eNOS and phospho-eNOS) was performed in HMEC-1 cells plated in 96-well format (Corning, NY, USA). Cells were washed twice with FBS-free medium and incubated with rabbit primary anti-NOS antibody (Thermo Fisher Scientific, Waltham, MO, USA) for 1 h. After washing, Alexa Fluor 594- or 488-conjugated goat-anti-rabbit secondary antibody (Jackson Immuno, Cambridgeshire, UK) was added for 30 min. DAF-FM Diacetate (Thermo Fisher Scientific, Hertfordshire, UK) was used to detect nitric oxide. Alternatively, cells after the treatment were stained with MitoTracker Deep Red FM (Thermo Fisher Scientific, Hertfordshire, UK) at final concentration of 0.5 μmol/L for 45 min, then washed twice with PBS and fixed in methanol. Cell nuclei were counterstained by DAPI. Images were taken and analyzed using fluorescence microscope (Zeiss, Dresden, Germany) and ZEN software v.3.3 blue edition (Zeiss, Dresden, Germany).