Orca er digital camera c4742 80

A total of 5 × 10⁴ neutrophils were seeded onto a 24-well flat-bottomed transparent plate in calcium- and magnesium-positive media supplemented with 3% mouse plasma and rested at 37° for 1 h, followed by stimulation for 2 h with 5 × 10⁵ CFU C. albicans (clinical isolate SC 5314) or media. After 2 h of incubation at 37°, NET formation was visualized by addition of the DNA stain Sytox (Life Technologies) (8.3 μM). Images were taken and analysed on a DM IRB (Leica) microscope with an Orca-ER Digital Camera C4742 80 (Hamamatsu) and an N PLAN PH1 lens (Leica), at a magnification of 10× and a numerical aperture of 0.25. Image acquisition software was Micromanager 1.4 and processing was performed using ImageJ 1.64. NETs were defined as Sytox⁺ areas > 2,000 μm².

Both HUVECs and EA.hy926 cells were isolated by centrifugation at 220 g for 3 minutes. The culture media surrounding the resulting cell pellet was removed and replaced by 1 ml of PBS with 1% BSA. Cells are then fixed using a 4% solution of formaldehyde (Thermo Scientific Product Number 28906). A 5-μl volume of Alexa Fluor® 594 anti-CD31 PECAM-1 (Biolegend Product Number 303126), Alexa Fluor® 488 anti-CD54 ICAM-1 (NovusBio Product Number DDX0150A488) and Alexa Fluor® 488 anti-CD62E E-selectin (Biorbyt Product Number orb170889) was added and then incubated on a rotational plate for a duration of 1 hour. Upon completion of the incubation period, the cells were then twice isolated by centrifugation at 220 g for 1 minute and washed in fresh PBS with 1% BSA. Cells were then loaded on to the biochip and imaged with an Olympus IX81 motorized inverted fluorescence microscope with an attached Hamamatsu ORCA - ER digital camera C4742-80 using a 10x and 20x objective.