Measuring EB1 Comet Length in GBM Cells

Cells were grown on 8-well chamber slides (Labtek, Thermo Fisher Scientific), precoated for 1 h with fibronectin (10 μg/ml) for U87-MG or with poly-DL-ornithine (Sigma-Aldrich) (10 µg/ml) for GBM6, to be treated for 6 h with ProA, digoxin, bufalin or digitoxin. As previously described^{16 (link)}, cells were incubated with the anti-EB1 (clone 5; BD Biosciences, San Jose, CA) and α-tubulin (clone DM1A; Sigma-Aldrich) primary antibodies, and then with Alexa488 or 568-conjugated secondary antibodies (Invitrogen). Staining was observed using either a Leica DM-IRBE microscope or a Leica TCS SP5 confocal laser-scanning microscope (Leica, Heidelberg, Germany). Images were acquired using Metamoph software or the Leica Confocal software, and were processed using Image J software. For each experimental condition, at least 100 EB1 comets (in at least 10 cells) were examined to measure their length.