Briefly, the Friedl laboratory provided HT1080 cells. Before sharing among the two groups, the Sahai laboratory validated this cell line by comparing its STR profile to the published ones. Mycoplasma infection was excluded prior to the experiments. Cells were cultured in T75 flask with 10% CO2 at 37°C. 3D spheroid culture and labeling were performed as follows:
Multicellular spheroids containing 1,000 HT1080 cells were generated using hanging‐drop culture method (Del Duca et al, 2004 (link)).
The spheroids were embedded in rat‐tail collagen I (Corning, Cat no. 354249), in up to 18 wells of 96‐well imaging plates per collagen concentration, using 1 spheroid per gel and a final collagen concentration of 2.5 or 6 mg/ml. Former protocols for spheroid embedding (Wolf et al, 2013 (link); Haeger et al, 2014 (link)) were adapted to have control over the number of spheroids per well, spheroid height with respect to imaging window and the onset of collagen polymerization, to minimize variation between technical repeats per plate.
Plates were incubated for 24 h at 37°C to establish cancer cell invasion in three dimensions, prior to fixation in 4% PFA.
The 3D cell cultures were fluorescently stained with DAPI (Sigma, D9542, 2 μg/ml) and AlexaFluor633‐Phalloidin (Molecular Probes, A22284, 1:200 dilution) and stored (preferably for < 48 h) at 4°C prior to imaging.
Hu J., Serra‐Picamal X., Bakker G., Van Troys M., Winograd‐Katz S., Ege N., Gong X., Didan Y., Grosheva I., Polansky O., Bakkali K., Van Hamme E., van Erp M., Vullings M., Weiss F., Clucas J., Dowbaj A.M., Sahai E., Ampe C., Geiger B., Friedl P., Bottai M, & Strömblad S. (2023). Multisite assessment of reproducibility in high‐content cell migration imaging data. Molecular Systems Biology, 19(6), e11490.
Other organizations :
Karolinska Institutet, Radboud University Nijmegen, Radboud University Medical Center, Ghent University, Weizmann Institute of Science, The Francis Crick Institute, VIB-UGent Center for Inflammation Research
Spheroid embedding (one spheroid per collagen gel)
Incubation time (24 h before fixation)
controls
No positive control specified
Mycoplasma infection was excluded prior to the experiments (negative control)
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