About 2.5 × 104 cells were seeded in chamber slides (Ibidi). After 24 h of incubation, the cells were transfected with pEGFP-C1 LifeAct-EGFP (Addgene 58470) using the K2® Transfection Reagent from Biontex according to the manufacturer’s instructions. After incubation for 24 h, time series were taken every 50 s for 17 frames by fluorescence microscopy at 100-fold magnification using the IXplore Live microscope imaging system from Olympus. Three filopodia per cell were measured by analysing its velocity (distance/time; µm/sec) using the Fiji software.
Ixplore live microscope imaging system
Manufactured by Olympus
The IXplore Live microscope imaging system is a versatile and advanced microscope designed for live cell imaging. It provides high-quality imaging capabilities, enabling users to observe and capture real-time cellular processes. The system features state-of-the-art optics, advanced imaging technologies, and a user-friendly interface to facilitate seamless operation.
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4 protocols using ixplore live microscope imaging system
1
Microscopic Analysis of Filopodia Dynamics
2
3D Spheroid Growth Dynamics
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Cells were grown in micro-molds (Merck, MicroTissues® 3D Petri Dish® micro-mold spheroid kit) as spheroids and fixed with type I collagen. Growth of the spheroids were assessed right after collagen I treatment and 24 h later by light microscopy at 4-fold magnification using the IXplore Live microscope imaging system from Olympus. The area covered by the spheroids were measured at time point 0 and 24 h, and growth was measured by calculating the ratio of these areas using the Fiji software.
3
EGFP-ITPKA Transfection and Imaging
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A total of 2.5 × 104 cells were seeded in chamber slides (Ibid). After 16 h of incubation, the cells were transfected with EGFP-ITPKA [4 (link)] or pEGFP-C1 LifeAct EGFP (Addgene 58470) using the K2® Transfection Reagent from Biontex according to the manufacturer’s instructions. After further incubation for 24 h, the cells were optionally fixed with 4% paraformaldehyde/4% sucrose, stained with Alexa568-coupled phalloidin, and analyzed by fluorescence microscopy using the IXplore Live microscope imaging system from Olympus.
4
Quantifying Collagen Invasion in 3D
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Type I collagen gels were prepared to a final concentration of 1 mg/ml and an invasion assay was performed as described [32 (link)]. In brief, collagen was spread onto wells of a 6-well plate and the collagen was incubated for 1 h in a humidified atmosphere of 5% CO2 at 37°C. Gels were seeded with 105 cells and incubated for 24 h. Sixteen fields of view were automatically captured at fixed points at 20-fold magnification by light microscopy and cells with invasive protrusions were counted (see representative images in Supplementary Figure S3). Invasion rate was expressed as percentage of invasive cells relative to all cells counted. To stain F-actin, gels were cut into small pieces and cells were fixed with 4% paraformaldehyde/4% sucrose and permeabilized with 0.5% Triton X-100 in PBS for 15 min. Thereafter, the gels were blocked with 2% BSA/1% glycine in PBS for 30 min and stained with Alexa488-coupled phalloidin for 30 min. Gels were mounted onto a slide and imaged at 100-fold magnification using the IXplore Live microscope imaging system from Olympus.
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