Primary Cultures for IHC-Viability Assays

Preparation of Primary Mesencephalic Cultures

The mesencephalic neurons and glia are dissociated from neuronal tissue with trypsin.

Note: Final concentration, 26 mg/ml in 0.9% [w/v] NaCl.

Note: 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 are treated for 48 h with AraC (20 mM) to inhibit the growth of glial cells.

Lentiviral Transductions of Primary Cultures

AraC-treated primary cultures are transduced with lentiviral particles in the presence of polybrene.

Note: The concentration of polybrene is 6 mg/ml. Control cells are incubated without lentivirus.

After a 72 h transduction period, the cells are treated with fresh media for an additional 48 h and analyzed immunocytochemically.

Immunocytochemistry of Primary Midbrain Cultures

Primary cells are fixed in 4% (w/v) paraformaldehyde in PBS for 30 min.
The cells are permeabilized and blocked simultaneously for 1 h 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 are treated overnight at 4°C with an anti-MAP2 monoclonal IgG and an anti-TH polyclonal antibody to monitor relative dopaminergic cell viability.
To determine the lentiviral transduction efficiency, un-transduced cells or cells transduced with lacZ lentivirus (expressing beta-galactosidase fused to the V5 epitope) are treated at RT for 1 h with:

After washing, the cells are treated for 1 h at RT with goat anti-mouse IgG conjugated to Alexa Fluor 488 and goat anti-rabbit IgG conjugated to Alexa Fluor 594.
The primary and secondary antibodies are prepared by diluting stock antibody solutions in PBS with 1% (w/v) BSA.
The coverslips are mounted onto slides using ProLong Gold Antifade reagent, dried at RT overnight. Sealed with clear nail polish.

Measurement of Primary Neuron Viability

MAP2- and TH-immunoreactive primary neurons can be counted in 10 randomly chosen observation fields for each experimental condition at a total magnification of 200x.
The data should be expressed as the percentage of MAP2-positive neurons that are also TH-positive.
Each experiment should be repeated 3-4 times using embryonic neurons isolated from independent animals.

Note: Statistical analyses can be carried out using the program GraphPad Prism®, Version 4.0.

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