Lab tek 2 chambered cover slides

Labteks were coated with poly-D-lysine (Sigma-Aldrich, #P6407, 0.5 mg/ml) for 1 h at room temperature for adherence of HEK-293-T cells; 1 × 10⁵ HEK-293-T cells were seeded at least 4 h before transfection on four-well Lab-Tek II chambered cover slides (Nunc, cat. no. 155409) and cultured at a 5% CO₂ atmosphere at 37°C.
HEK-293-T cells (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; #ACC635) were maintained in T25-culture flasks (Thermo Fisher, Cat. Nr. 156340) in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, #D5796) supplemented with 10% FCS (Sigma-Aldrich, #F7524), and 1% Pen-Strep (Sigma-Aldrich, #P4333).
All experiments with primary neuronal cultures carried out in accordance with the guidelines established by the European Communities Council (Directive 2010/63/EU of September 22, 2010) were permitted by the Italian Ministry of Health and followed the rules approved by the Italian Institute of Technology. Primary cultures of hippocampal neurons were prepared from P0-P1 C57BL/6J mice as previously published (Polenghi et al., 2020 (link)). Neurons were plated at a density of 70 × 10³ cells/cm² on poly-D-lysine pre-coated coverslips and kept in Neurobasal-A medium (Thermo Fisher, Italy) supplemented with B-27 (Thermo Fisher, Italy) 2%, Glutamax 1% (Thermo Fisher, Italy), and gentamycin 5 mg/ml (Sigma) at 37°C in 7.4% CO₂.

HeLa cells and COS-7 cells were cultured at 37 °C and 5% CO₂ using DMEM high glucose with pyruvate (4.5 g l⁻¹ glucose, with GlutaMAX^TM supplement) supplemented with 10% fetal bovine serum and 1 × penicillin–streptomycin (all gibco^®, ThermoFisher Scientific) or DMEM high glucose w/o phenol red (4.5 g l⁻¹ glucose) supplemented with 4 mM l-gluthamine, 10% fetal bovine serum and 1 × penicillin–streptomycin (all gibco^®, ThermoFisher Scientific). HeLa cells were from ATCC (ATCC® CCL-2™) and COS-7 cells were a kind gift of the Manley lab (LEB, EPFL).
Cells were seeded in Lab-tek^® II chambered cover slides (nunc) 1–2 days before fixation in DMEM high glucose w/o phenol red (4.5 g l⁻¹ glucose) supplemented with 4 mM l-gluthamine, 10% fetal bovine serum and 1 × penicillin–streptomycin (all gibco^®, ThermoFisher Scientific).