Fcs2 live cell chamber

Live cell imaging was performed in an environmentally controlled FCS2 live cell chamber (37°C temp and 5% CO₂) (Bioptechs, Butler, PA) system using a confocal microscope (Nikon A1, Nikon) controlled by NIS-Elements software. Prior to imaging, 2×10⁵ cells were plated on 35 mm glass bottom microwell dish (Mat-Tek, Ashland, MA) and incubated for 24 hours. Cells were then incubated for 2-3 minutes with Hoechst 33342 in complete media to label nuclei. The dishes were then immediately assembled into a heated chamber and time-lapse imaging was initiated immediately. The Z stacks images of selected cells were acquired every 5 minutes using excitation at 488 nm (for GFP) and 403.8 nm (for Hoechst 33342). Fluorescence was captured through an Plan Apo VC 60× oil immersion objective, NA = 1.40 (Nikon) by using perfect focus system to correct possible focus drift during time lapse imaging. Culture medium containing BITC was used, and the temperature of the chamber was maintained at 37°C during the imaging. GFP excitation was kept as low as possible to avoid photo-destruction of the cell. For all studies, 3-6 fields per dish and duplicate dishes per condition were evaluated in three independent experiments. Images were processed using MetaMorph (Molecular Devices, Sunnyvale, CA), and Adobe Photoshop.