Imagexpress micro xl system

Microscopy was conducted using a 60× objective lens and an ImageXpress Micro XL system (Molecular Device, Sunnyvale, CA, USA).

A blue fluorescence protein (BFP) cassette was amplified from the pFA6a-link-yomTagBFP2-Kan plasmid and integrated into the genome to generate an Hsp104-BFP fusion protein in all tested strains [77 (link)]. Log-phase cells growing in YPD at 25°C were transferred to a 37°C water bath and cells were collected at 0, 1, 2, and 3 hours after the temperature shift. The samples were resuspended in 200 μL PBS and transferred to a Glass Bottom ViewPlate-96F (PerkinElmer, Waltham, MA) that was precoated with concanavalin A (cat. no. C2010, Sigma-Aldrich). Images of Hsp104-BFP foci were acquired using an ImageXpress Micro XL system (Molecular Devices, San Jose, CA) and analyzed using a custom-built module under MetaXpress High-Content Image Acquisition and Analysis Software (Molecular Devices).

U-251, LN-229, and U-87 cells were seeded in a 96-well plate (at a concentration of 25 × 10⁴ cells/0.32 cm²) and maintained in a 5% CO2 humidified atmosphere at 37 °C to form a monolayer with confluence. Immediately after irradiation, the cell monolayer was scratched in a straight line with a 200 μL sterile micropipette tip and washed with 1XPBS (pH = 7.4) 3 times to remove cell debris. Cells were incubated with complete culture medium for another 96 h. The initial areas of the wounds were imaged at time zero (t = 0 h) and after scratching at 24 h, 48 h, 72 h, and 96 h (t = ∆ h) using the ImageXpress Micro XL System (Molecular Devices LLC, San Jose, CA, USA). The percentage of migratory ability was assessed using the following equation:
where at = 0 h is the area of wound calculated immediately after wounding, and at = Δ h is the wound healing area calculated at 24 h, 48 h, 72 h, and 96 h after wounding.