Axio observer microscopy

Migratory capacity of cells on the ECM components fibronectin, and type-I collagen was examined using IBIDI chambers in triplicates. Briefly, uncoated IBIDI dishes (ref #81151) were coated overnight at 4°C with type-1 collagen (Sigma-Aldrich) or fibronectin (Sigma-Aldrich). Then, a culture insert (IBIDI, ref #80206) was used to form chambers for cell seeding. After removal of the separation wall, cell migration was monitored under time lapse microscopy using Zeiss Axio Observer Microscopy. Migration distance of each cell line was quantified at different points (n = 6) using Image J. Results were obtained after 16 h migration for all the substrates. Cell migration was also monitored using standard tissue culture dishes suitable for wound healing migration assays (IBIDI, ref #81176) following manufacturer instructions. Proliferation rate was calculated by direct cell counting on four different fields over a 4 days period.

For focal adhesion identification, 2 × 10³ cells/well were seeded and left for 24 h to adhere to each surface. Cells were fixed in paraformaldehyde 4%, permeabilized with Triton 0.1%, blocked with bovine serum albumin (BSA) 1% and incubated overnight with anti-vinculin antibody diluted 1:150. Cells were PBS washed and incubated with secondary antibody conjugated to Alexa-Fluor 488 (1:500) for 1 h. Fixed cells were also co-incubated with Alexa-Fluor 546 conjugated phalloidin for 30 min (1:50) to stain actin fibers and mounted on glass slides with 4′,6-diamidino-2-phenylindole (DAPI) Fluorshield to stain nucleic acids in the nucleus. Stained cells were digitally registered using the fluorescent filters for vinculin (green), actin fibers (red) and nuclei (blue stains). The Axio Observer microscopy and Z-stack accessories were used to acquire images and AxioVision 4.8.1 (Zeiss, Oberkochen, Germany) software was used for image processing and overlay.

Cellular uptake of the PEI-Apt-siRNA nanocomplex was performed in MCF-7 and WERI-Rb1 cells using flow cytometry (BD Science FACS Caliber). The 200 nM of aptamer alone or PEI-Apt-siRNA nanocomplex (200 nM EpApt and 200 nM siRNA) were incubated with 2 × 10⁵ cells for 4 h followed by washing with 1× PBS two times and the cells were analyzed using BD FACS Calibur. The unstained cells and the scrambled aptamer treated cells were included as controls. The uptake of the aptamer alone, and PEI-Apt-siRNA nanocomplex was visualized using fluorescent Axio Observer microscopy (Zeiss, Germany).