Cells were maintained in serum-free medium for 8 h/d, and the culture medium was collected. After the culture medium was collected seven times, the combined culture media were concentrated with a filtering device (Biomax-10k NMWL; Millipore, Bedford, MA) by centrifugation. Cultured cells were lysed in lysis buffer (137 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, [pH 8.0], containing a protease inhibitor [Roche, Basel, Switzerland]). The cell lysates were sonicated and disrupted by the freeze–thaw method four times. The cell lysates and culture medium were centrifuged, and the supernatant was used for each analysis. The expression of BDNF in the lysates was measured by an immunoassay system (Emax; Promega).