Eclipse ti e n storm microscope

Freshly isolated peripheral blood from MYH9-RD patients and from healthy individuals was used for neutrophil isolation using Polymorphprep (Axis-Shield) and contaminating erythrocytes were lysed using 155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA. Neutrophils were resuspended in Hank's balanced salt solution without calcium and magnesium and adhered to poly-D-lysine-coated cover glasses for 15 min or transferred to 24 well plates with poly-D-lysine-coated cover glasses and incubated with 10 μM oleic acid for 1 h at 37 °C. Neutrophils were fixed with 4% paraformaldehyde and subjected to NMIIa or LD stainings. Neutrophils were imaged with a Nikon Eclipse Ti-E N-STORM microscope, equipped with a 100 × Apo TIRF oil objective NA 1.49. LD images were automatically deconvolved using Huygens software (Scientific Volume Imaging). Maximum intensity projections were generated using MatLAB and thresholded LD images were quantified using the ImageJ analyse particles tool. The MYH9-RD patients provided a written informed consent in accordance with the Declaration of Helsinki.

DAF-2 DA, fluorescent nitric oxide probe (ab145283, Abcam, UK) with a final concentration of 10 µM was used to evaluate nitric oxide in the cells. This probe begins to display fluorescence when the nitric oxide generates in the cells. After 24 h of cell growth in the reservoir on the microchip, DAF-2DA was mixed with 800 µL fresh culture media and incubated in the dark for 20 min before the experiment. In the same manner as in hypoxia experiments, Nikon Eclipse Ti-EN- STORM microscope used for nitric oxide imaging and light was turned off before each 15 min interval. To evaluate the nitric oxide response from one or two channels, data were collected from two individual experiments (n = 2).