Zen 2009 image analysis software

TALL-104 cells suspended in complete Iscove's modified Dulbecco's medium at 1 × 10⁶ cells/ml were treated with appropriate compounds (TPA, TG) in a manner analogous to flow cytometry experiments described in FRET experimental design. Next cell aliquots were removed and immediately fixed in ice-cold 2% paraformaldehyde before cell activation and 3 and 6 min after the addition of TPA/TG mixture and stored at 4°C overnight. The next day, cells were washed with ice-cold PBS and stained with primary labeled (phycoerythrin) fluorescent mAbs (mouse anti-human CD11a/LFA-1α, clone HI111 [PE] from BD Biosciences, San Jose, CA) for 30–40 min on ice according to manufacturer's instructions. Samples were mounted using SlowFade Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (Life Technologies). Fluorescence microscopy was performed with a confocal microscope (Zeiss 510 Laser Scanning Microscope 510-META) using an oil immersion objective lens at the University of New Mexico Fluorescence Microscopy and Cell Imaging Shared Resource facility. Images were obtained and processed using ZEN 2009 image analysis software (Carl Zeiss MicroImaging).