The mesencephalic neurons and glia are dissociated from neuronal tissue with trypsin (final concentration, 26 mg/ml in 0.9% [w/v] NaCl)
Cells are plated on coverslips previously treated with poly-L-lysine (5 mg/ml).
The media consists of DMEM, 10% (v/v) FBS, 10% (v/v) HS, penicillin (100 U/ml), and streptomycin (100 mg/ml).
Four days later, the cells were treated for 48 hr with AraC (20 mM) to inhibit the growth of glial cells.
B. Lentiviral transductions of primary cultures
AraC-treated primary cultures are transduced with lentiviral particles in the presence of polybrene (6 mg/ml).
Control cells were incubated without lentivirus.
After a 72 hr transduction period, the cells were treated with fresh media for an additional 48 hr and analyzed immunocytochemically.
C. Immunocytochemistry of primary midbrain cultures
Primary cells were fixed in 4% (w/v) paraformaldehyde in PBS for 30 min.
The cells were permeabilized and blocked simultaneously for 1 hr with PBS containing 1% (w/v) BSA, 10% (v/v) FBS, and 0.3% (v/v) Triton X-100.
After washing with PBS, the cells were treated overnight at 4°C with an anti-MAP2 monoclonal IgG (1:500) and an anti-TH polyclonal antibody (1:500) to monitor relative dopaminergic cell viability.
To determine the lentiviral transduction efficiency, untransduced cells or cells transduced with lacZ lentivirus (expressing beta-galactosidase fused to the V5 epitope) are treated at room temperature for 1 hr with: