Primary cultures and their survival were established and investigated as described previously15 (link). E13.5 embryos of C57Bl/6J wild type mice (6 months old) were dissected according to Planket et al.16 (link). 30 000 live cells were plated on 96-well plate pre-coated with poly-L-ornithine in culture medium (Neurobasal Medium + B27 supplement 1x + 2 mM L-Glutamine) and placed in the incubator. After 45 minutes required for cells attached, 100 µl of medium containing vehicle or investigated drugs were added. Drugs being the subject of investigation: GDNF growth factor – a positive control (100 ng/µl; PeproTech), reboxetine (10 µM; Tocris, #1982) and phenylephrine (100 µM; Sigma, P6126). The optimal dosage of investigated pharmaceuticals was chosen upon own experience and available literature17 . 48 hours after plating half of the medium was exchanged with fresh one containing the respective drug concentrations. 5 days after plating cells were fixed with 4% PFA, immunostained with anti-TH antibody (Millipore, AB1542, 1:2000) and counterstained with DAPI. The plates where imaged by an inverted fluorescent microscope (AxioObserver, Carl Zeiss, Germany) at 5x magnification and the number of surviving TH+ cells in each separate well were counted automatically in an unbiased way with use of the ImageJ software18 (link).
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