Petri dishes

Dissociated cortical neurons were prepared and maintained as previously described for hippocampus neurons (Aakalu et al., 2001 (link)). Cortices from postnatal day one old rat pups of either sex (RRID:RGD_734476; strain Sprague-Dawley) were dissected, dissociated by incubating with L-cysteine-papain solution at 37°C and plated onto 10 cm Petri dishes (MatTek, Ashland, MA) previously coated with poly-D-lysine. Cultured cells were kept in Neurobasal-A medium (Invitrogen, Carlsbad, CA) supplemented with B-27 (Invitrogen) and Glutamax (Invitrogen) at 37°C and 5% CO₂ for 19-20 days.
All experiments complied with national animal care guidelines and the guidelines issued by the Max Planck Society and were approved by local authorities.

MatTek Petri dishes were coated with poly-ornithine (100μg/mL) and laminin (5μg/mL) according to manufacturer’s recommendations. Cortices from E12.5 NMRI mouse embryos were dissected (around 14 cortices for 10mL of culture medium) and mechanically dissociated. The cell suspension was filtered using a strainer of 40μL and 800μL of cell suspension were added to the coverslip of MatTek dishes. After 2h, 1mL of DMEM-F12 medium supplemented with B27 containing vitamin A (1:50) penicillin/streptomycin (1:100), L-glutamine (1:100), EGF and bFGF (20ng/mL final). Cells were kept in a humidified incubator saturated with 5% CO₂ for 24h. MGEs were microdissected and cut into 4 homogeneous pieces. Twenty pieces of MGE were plated in contact with cortical progenitors or on top of a Millicell insert and maintained in culture for a an additional period of 24h. After this period, 1.5 μL of 10mM EdU were added to each dish for a period of 30 min.

Three extracellular substrates were tested for their ability to support cell viability and to modulate the morphology of individual cells and cell clusters. Glass bottom 47 mm diameter Petri dishes (MatTek, Ashland, MA, USA) were coated with 300 µL of 100 µg/mL Poly-L-lysine (PLL, Sigma-Aldrich), 15 µg/mL Concanavalin A (Con-A, eBioscience) or 80 µg/mL Laminin (LM; Gibco, Grand Island, NY, USA) at room temperature (25 °C) for 1 h. Uncoated dishes served as controls. The supernatant was then removed, and the dishes were washed 3× with PBS. All liquid was removed from the dish and placed in a 25 °C incubator overnight. Cell cultures were maintained in a 25 °C incubator and fresh Schneider’s medium was added to cell cultures after 24 h. Cells were assessed for morphology and viability at 0, 24 and 48 h. Brightfield images of cell cultures were taken to assess morphology of individual cells and RPC clusters. Viable cells on each substrate were tested after 24 h and 48 h using the Colorimetric Cell Viability Kit III XTT (Invitrogen). Potential reductions in cell viability over time were assessed by comparing XTT absorbances with values obtained from assays of samples of newly-dissected cells (n > 15 eye-brain complexes, isolated as described). All absorbance values were normalized against those on uncoated Petri dishes.

Cells of the three tumor cell lines were grown on Petri dishes with a glass bottom (MatTek Corp., Ashland, MA, USA). Then, they were incubated with the medium containing compounds 5a and 5b at the concentration of 2.5 μM for 24 h in the dark. The cells had been washed twice with PBS prior to imaging of the subcellular localization patterns of the fluorescent sensitizers with an Axiovert 40CFL microscope (Carl Zeiss, Jena, Germany) using an oil immersion objective (100 × NA 1.25). A band-pass of 300–400 nm excitation filter was used to detect the PpIX derivatives with a long-pass of 630 nm emission filter.