Leica tcs sp5 confocal laser scanning microscope

ARPE-19 cells were seeded in a 4-well chamber slide (Cat#PEZGS0496, EMD Millipore, Billerica, MA) and cultured for 24 hours to 80–90% confluence. Cells were subsequently incubated with 5 × 10⁷/ml of 1 µm-diameter Fluoresbrite® YG Carboxylate Microspheres (Cat#15702, Polysciences, Warrington, PA, US) for 6 hours. The cells were then washed with DPBS (Cat# BE17-515Q, Lonza, Walkersville, MD, US) three times, after which 4% paraformaldehyde (Cat# 28906, Thermo Fisher Scientific, Rockford, IL, US) was used to fix the cells for five minutes. Then, the cells were washed with DPBS 3 times (5 minutes for each wash). Finally, cells were stained with DAPI (diamidino-2-phenylindole, Cat# 62248, Thermo Fisher Scientific, Germany) for 2 minutes. After a brief rinse with DPBS, cells were mounted under a coverslip with Vectashield mounting medium (Cat# H-1000, Vector Laboratories, Burlingame, CA, US). The cell slide was observed and imaged using Leica TCS SP5 laser-scanning confocal microscope (Leica, Wetzlar, Germany). A Z-stack of 50 optical sections was collected from the bottom of the culture chamber toward the top of the slide to include the entire cell layer. The Leica Application Suite (Version 2.6.0) software was used to collect the images. The final images were assembled with Image J (Version 2.0.0).

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.

Cells were dissociated into single cell suspensions using trypsin and seeded onto cover slips in a 24-well plate. Cells were fixed by incubation with 4% paraformaldehyde for 15 min at room temperature after each experiment. Cells were permeabilized with 0.3% Triton X-100 for 15 min at room temperature and then blocked by incubation with 10% bovine serum albumin for 1 h at 37°C. Coverslips were then incubated at 4°C with primary antibody solution overnight, washed three times in PBS, and incubated with secondary antibody solution containing Hoechst 33342 for 1 h at 37°C. Secondary antibodies used were: Alexa Fluor®-488 and −594 goat anti-rabbit or anti-mouse IgG (ZSGB-BIO, China). Finally, coverslips were washed three times and mounted on microscope slides using mounting medium (ZSGB-BIO). Images were captured under a Leica TCS SP5 confocal laser-scanning microscope (Leica, IL, USA), with co-localization analysis and image merge conducted using Leica software according to the recommended procedures.