Migration assay was adapted from previously described protocols [21 (link),22 (link)]. In more detail, spheroids were transfected with different CPP/siRNA NPs 4 days after seeding. Then, 48 h after transfection, spheroids were transferred to the middle of Nunc Lab-Tek 8-well coverslips (Thermo Fisher Scientific, Waltham, MA, USA) previously coated with 0.1% gelatin (Naxo, Tartu, Estonia), one spheroid per well. A further 3 days after that, 3D cultures were fixed with 4% formaldehyde (Naxo, Tartu, Estonia) and stained with 1% methylene blue (BioGnost, Zagreb, Croatia) and 1% eosin (BioGnost, Zagreb, Croatia). Spheroids were visualized and photographed under stereomicroscope (Leica M165FC, Wetzlar, Germany). Images were analyzed and cell migration surface area was measured using ImageJ software.
Migration evaluation was the only assay, where HT1080 cells were used instead of U-87MG due to technical reasons. Namely, U-87MG cell migration from the spheroids was scattered and irregular, which did not allow the quantification of the results. Therefore, the U-87MG cell line was replaced with another 3D model available that had previously shown to exhibit regular cell migration patterns according to our experience.
Migration evaluation was the only assay, where HT1080 cells were used instead of U-87MG due to technical reasons. Namely, U-87MG cell migration from the spheroids was scattered and irregular, which did not allow the quantification of the results. Therefore, the U-87MG cell line was replaced with another 3D model available that had previously shown to exhibit regular cell migration patterns according to our experience.
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