NGF and Anti-NGF Antibody Treatment Protocol

For the treatment with 100 ng/mL NGF or 100 ng/mL anti–NGF neutralizing antibody (RnD Systems, Minneapolis, MN, USA), 1.5 mL/well cell suspension of 6.7 × 10⁴ cells/mL were plated in 12-well plates (Falcon^TM, Durham, NC, USA) in serum-free, albumin containing medium [43 (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, MO, USA) in serum-free albumin-containing medium for 30 min at 37 °C ensuring cell cycle arrest [42 (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 100 ng/mL recombinant human NGF [45 (link)] or with 100 ng/mL anti NGF neutralizing antibody (RnD Systems) 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).