Fig. 2
BDNF levels in various culture media and the protein’s effect on cell migration using the MDA-MB-231 line. Different breast cancer cell lines, such as MCF-7, BT-474, SK-BR-3, MDA-MB-468 and MDA-MB-231 (4 × 105/well), were cultured for 1 day and this was followed by replacement by low serum medium at 24 h (white bar), 48 h (grey bar) and confluence (black bar). Next the culture media were collected to allow the BDNF level in each medium (a) to be measured by ELISA as described in Methods. A cell migration assay (b) using a cell culture insert, as described in Methods, was carried out in the presence of serum free medium (the negative control, NC), 10% FBS medium (positive control, PC) BDNF (50 ng/mL), or 2-day-cultured medium (conditioned) medium (CM) using MDA-MB-231 cells without or with pretreatment with a neutralization antibody against BDNF or non-specific antibodies. The percentage of migratory cells (c) was measured by comparing the treated group with the vehicle group. Data are expressed as mean + SEM. *, p < 0.05 compared to fresh prepared medium (day 0) or serum free (negative control), one way ANOVA. #, p < 0.05 compared to CM-treated group (n = three or four independent experiments in each group) by Mann-Whitney U test