Nunc lab tek 2 8 well glass chamber slides

To generate mouse cortical neurons, Cdkl5^+/– female and Cdkl5^+/Y male or Cdkl5^+/– female and Cdkl5^+/Y/GFP-LC3^+/+ male mice were mated and the brains from littermate offspring harvested on embryonic day 15 (E15). The cortex was dissected from the rest of the brain under a dissecting microscope. Cortical cells were dissociated in 2.5% Trypsin (Thermo Fisher Scientific) and DNase I (Sigma-Aldrich), and resuspended in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were then plated on Nunc Lab-Tek II 8-well glass chamber slides (Thermo Fisher Scientific), or 12-well dishes coated overnight with 0.1% polyethyleneimine from Sigma-Aldrich. After 2 hours, the medium was replaced with Neurobasal medium supplemented with B27 (Invitrogen), 2 mM glutamine (Thermo Fisher Scientific), and 5 μM cytarabine (AraC) (Sigma-Aldrich) for 24 hours to suppress growth of non-neuronal cells, at which point fresh medium without AraC was added to culture neurons for 7 days.

HeLa cells (1 × 10⁴) were cultured for 48 h at 37°C in Nunc Lab-Tek II 8-well glass Chamber Slides (Thermo Fisher Scientific, Waltham, MA). Cells were then fixed with 4% paraformaldehyde for 10 min at room temperature followed by two washes in PBS(-). Fixed cells were permeabilized for 20 min at room temperature with 0.5% Triton X-100 in PBS(-) prior to incubation with antibodies. For staining, cells were incubated at 4°C overnight with primary antibodies diluted appropriately in 20% ImmunoBlock (DS Pharma Biomedical Co., Ltd., Osaka, Japan)/PBS(-). Cells were washed twice with PBS(-) and incubated with Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA) and Hoechst33342 (Thermo Fisher Scientific, Waltham, MA) for 2 h at room temperature. Samples were then washed twice with PBS(-) and mounted with VECTASHIELD (Vector Laboratories, inc., Burlingame, CA). Images were acquired by FV10i Confocal Laser Scanning Microscopy (OLYMPUS Corp., Tokyo, Japan)