Orca flash4.0 lt digital cmos camera c11440 42u

The effect of β-escin on sprouting efficiency of HUVECs was assessed in the in vitro fibrin gel bead assay as described previously [49 (link)]. Briefly, 2500 Cytodex beads (GE Healthcare, Chicago, IL, USA) were incubated with 106 HUVECs (5 h, 37 °C, and 5% CO₂) and plated overnight on a 10 cm dish to remove unattached cells. Next day, the cell-covered beads were resuspended to a concentration of ~350/mL in 3 mg/mL fibrinogen (Sigma-Aldrich) solution containing 0.15 U/mL aprotinin (Sigma-Aldrich), 30 ng/mL bFGF, and 25 ng/mL VEGF. Aliquots were mixed with thrombin (Sigma; 0.625 U/mL), distributed in 12-well plates (150 mL/well), and left to clot for 5–10 min. After further incubation (10 min, 37 °C, and 5% CO₂), medium containing 30 ng/mL bFGF and 25 ng/mL VEGF was added to cover the clot. Sprout formation was examined microscopically and photographs were taken using an Olympus IX70 inverted light microscope equipped with a cooled CCD camera (Hamamatsu ORCA-Flash4.0 LT Digital CMOS camera C11440-42U) and analyzed using ImageJ network analysis software (Bethesda, MD, USA).

Image acquisition was performed via custom microscope equipped with a scientific CMOS (sCMOS) camera (ORCA-Flash4.0 LT Digital CMOS camera C11440-42U; Hamamatsu , Boston, MA). Fluorescence excitation is accomplished with a 5W LED (LZ1-00B200, 460 nm; LedEngin, San Jose CA). Image optics included a Leica N Plan 10 × 0.25 PH1 microscope objective lens, an excitation filter (HQ 470/50), dichroic mirror (FF506-Di02), emission filter (FF01-536/40), and a commercial SLR lens focused to infinity as the tube lens (Nikon Zoom-NIKKOR 80–200 mm f/4 AI-s). Hippocampal imaging data were collected and processed using a computer equipped with dual Intel Xeon processors, 128 GB RAM, and a GeForce GTX Titan video card. The use of the graphics card allowed for images to be processed offline by the GPU and therefore did not dependent on substantial amounts of available computer RAM. Images were captured as multi-page tagged image file format (mpTIFF) using the default image-capture software bundled with the purchase of a sCMOS camera from Hamamatsu (HC Image Live; Hamamatsu; Boston, MA).

Endothelial cells were seeded and grown to confluence in 6-wells plates and then treated with β-escin (60 µg/mL) in cM199 medium. Pictures of the monolayers were taken using an inverted light microscope (Olympus IX70, Tokyo, Japan) equipped with cooled CCD camera (Hamamatsu ORCA-Flash4.0 LT Digital CMOS camera C11440-42U, Hamamatsu, Japan) at the time points 24 h, 48 h and 72 h and the number of cells were counted.