Ix51 microscope

Migration assays were performed using 24-well Boyden chambers (8 µm pores, BD Biosciences) as we previously described [25] (link). The cells were plated at a density of 1×10⁵ cells/100 µl of medium in the inserts, and incubated for 3 h at 37°C, followed by staining using a Diff-Quick kit (SIMENS). Pictures were taken with a microscope to count the number of migrated cells. The scratch wound method was also employed in some experiments as follows. Cells were seeded and allowed to form a monolayer for 24 h. The dish was scraped with the tip of a 100 µl pipette, and the resulting wound was washed with PBS two times [12] (link). Culture medium containing 10% FBS was added to the cells, and the cells were incubated at 37°C in 5% CO₂. Bright-field images were captured (Olympus IX51 microscope) and analyzed (Adobe Photoshop). The total number of pixels in empty spaces inside the wound were counted and normalized to the control.

Live/dead and CyQuant viability assays were performed as previously described (12 (link)). For live/dead assays, hBMECs were costained with calcein-AM (live/green, 3 μM; Invitrogen) and propidium iodide (dead/red, 2.5 μM; Calbiochem). calcein-AM-positive versus PI-positive cells were resolved using an Olympus IX51 microscope and overlaid using Adobe Photoshop. hBMECs were incubated with CyQuant-NF (Thermo Fisher), and fluorescence was quantified using a BioTek FLx800 fluorimeter.