FiloMap (https://github.com/guijacquemet/FiloMAP) is an image analysis pipeline that can be used to map the localisation of proteins within filopodia from microscopy images. This Zenodo archive is composed of two-part: a test dataset that can be used to play with FiloMap. Example results produced by FiloMap when analysing this test dataset U2OS cells transiently expressing a VASP-GFP and MYO10-mScarlet (Jacquemet et al., 2019) were plated on fibronectin-coated glass-bottom dishes for two hours. Samples were fixed and permeabilised simultaneously using a solution of 4% (wt/vol) PFA and 0.25% (vol/vol) Triton X-100 for 10 min. Cells were then washed with PBS, quenched using a solution of 1 M glycine for 30 min, and incubated with SiR-actin (100 nM in PBS) at 4 C until imaging (minimum length of staining, overnight at 4 C; maximum duration, one week). Before imaging, samples were washed three times in PBS and mounted in vectashield (Vectorlabs). Images were acquired using a DeltaVision OMX v4 (GE Healthcare Life Sciences) fitted with a 60x 3 Plan-Apochromat objective lens, 1.42 NA (immersion oil RI of 1.516) used in SIM illumination mode (five phases and 3 three rotations). Emitted light was collected on a front-illuminated pco.edge sCMOS (pixel size 6.5 mm, readout speed 95 MHz; PCO AG) controlled by SoftWorx.

2022