2.8. Transduction of LifeAct GFP into HT1080 Cells and Life Cell Imaging

The lentiviral pLenti-LifeAct-GFP construct (a kind gift from Dr. S. Huveneers, AMC, Amsterdam, Netherlands) was packaged into lentivirus in HEK-293T cells, using second-generation lentiviral packaging plasmids, pMDG.2 and psPAX2 (both are kind gifts from Dr. D. Trono, Addgene plasmids #12259 and #12260), with GeneJammer transfection reagents (Agilent, Waldbronn, Germany). Lentivirus containing supernatant was harvested 72 h after transfection, concentrated with Lenti-X concentrator (Clontech, Saint-Germain-en-Laye, France) and used to transduce HT1080 cells with 10 µg/mL polybrene. Cells were selected with 1 µg/mL puromycin (Toku-E, Sint-Denijs-Westrem, Belgium). After 72 h, cells were seeded to 8-well plastic µ-slide (ibidi) coated with laminin-111 or collagen I (each at 10 µg/mL) and incubated at 37 °C and 5% CO₂ for 1 h. Adhesion and spreading were recorded with video confocal microscopy in a LSM800, at 37 °C, with 10× and 40× immersion oil objectives, with one z-stack picture/min for 10 min. For each condition, at least 30 cells were imaged.