BDNF and TGF-β1 Effects on Cell Proliferation

For the treatment with 0–50 ng/mL BDNF [13 (link)] or with 1 ng/mL TGF-β1 [20 (link)] (RnD Systems, Minneapolis, MN, USA), 6.7 × 10⁴ cells/mL were plated in serum containing medium [20 (link)] and cultured for 72 h. After that, the cells were washed with PBS and incubated with 10 µg/mL Mitomycin C (Sigma–Aldrich^®, St. Louis, MI, USA) in serum-free albumin-containing medium for 30 min at 37 °C ensuring cell cycle arrest [40 (link)]. Then the cells were washed twice with albumin-containing medium and subsequently treated with albumin-containing medium for two times 48 h supplied with 25 ng/mL recombinant human BDNF [13 (link)] for altogether 96 h. After completion of treatments, the cells were used for cell counting with trypan blue staining (Sigma, Darmstadt, Germany) in a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). One-thousand cells from all samples were plated in 75-cm² cell culture flasks grown in serum-supplemented medium for 3 weeks and the growing colonies were stained with gentian violet and were counted. The number of growing colonies were related to plated cell numbers (1000) [57 (link)].
During the treatments cells were filmed by a Juli BR live cell imaging system (Peqlab, Erlangen, Germany), which was also used for estimation of the percentage of covered area in the culture dish.