Penicillin streptomycin

PC12 cell line was obtained from Pasteur Institute of Iran (Tehran, Iran), and cultured on poly‐l‐ornithine (Sigma, USA) and laminin (Sigma)‐coated dishes in high‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% (v/v) heat‐inactivated horse serum (Sigma), 5% (v/v) heat‐inactivated fetal bovine serum (Gibco), and 100 U/ml penicillin–streptomycin (Gibco) at 37°C under a humidified atmosphere of 5% CO₂. To induce differentiation, cells were treated for 7 days in medium containing 50 ng/ml of NGF‐β (Cell Guidance Systems, USA), 100 U/ml penicillin/streptomycin and 1% (v/v) horse serum. The half volume of differentiating medium was refreshed every 2 days.

Twelve-week-old wild-type and Ecsit^N209I/N209I animals were sacrificed by cervical dislocation and bone marrow flushed from femur and tibia with PBS containing 0.6 mM EDTA. Cell suspension was filtered through a pre-wetted 70 µm cell strainer. Cells were pelleted at 400 g for 7 min (4°C). RBCs were removed by resuspending the pellet in 3 mL of RBC lysis buffer (155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA) and incubating for 1 min before diluting in 10 mL of PBS (0.6 mM EDTA) and again pelleted at 400 g.
Finally, cells were plated at a concentration of 2.5 × 10⁶ cells/mL in Dulbecco’s modified Eagle’s medium (DMEM; pyruvate, glutamine) containing 10% FBS, 100 U/mL penicillin–streptomycin and 100 ng/mL of macrophage colony-stimulating factor (MCSF; Cell Guidance Systems). Cells were maintained at 37°C, 5% CO₂ with media changes on Days 3 and 6. On Day 7, cells were harvested by manual scraping with PBS (0.6 mM EDTA). Cells were counted again and re-plated at a concentration of 6 × 10⁶ cells/well of a six-well plate in DMEM (pyruvate, glutamine), 10% FBS, 100 U/mL penicillin–streptomycin, 100 ng/mL MCSF. Plated cells were activated with 100 ng/mL of lipopolysaccharide (LPS) added directly to the media and incubated for 24 h at 37°C, 5% CO₂.
Activated cells were harvested by manual scraping with PBS (0.6 mM EDTA).