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Live-cell imaging

RT Ryouhei Tsutsumi
JH Jana Harizanova
RS Rabea Stockert
KS Katrin Schröder
PB Philippe I. H. Bastiaens
BN Benjamin G. Neel
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Expression vectors for HyPer3 or its mutants were transfected into Swiss 3T3 fibroblasts using Lipofectamine 3000 (Thermo Fisher), as per the manufacturer’s protocol. Twelve hours after transfection, cells were re-seeded (1 x 104 cells/well) into glass-bottom 8-well lab-tech chambers (Nunc) or µ-Slide 8 Well (ibidi), and then serum-starved in phenol red-free DMEM for 16 h. The fluorescence intensities of HyPer3-expressing cells were monitored by using an LSM700 confocal microscopy system with a stage-top incubator at 37 °C in a 5% CO2 atmosphere. Excitation was at 488 nm, and emission was monitored at 500–1000 nm. Images were obtained every 1 min, with PDGF added after the third scan. Cells that showed shrinking or detachment during the time-lapse were excluded from the analyses.

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