Sh sy5y cells

Human neuroblastoma SH-SY5Y cells were supplied by the Korean Cell Line Bank (Seoul, Korea). Cells were maintained in a minimum essential medium containing Earle’s balanced salts (HyClone, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (Life Technologies Corp., Carlsbad, CA, USA), 100 μg/mL streptomycin, and 100 U/mL penicillin at 37 °C in an atmosphere of 95% air and 5% CO₂.

Immunoblot was performed to confirm the intracellular signaling pathway through the BDNF–TrkB pathway. To induce differentiation of SH-SY5Y cells (Korea Cell Line Bank), 10 μM of retinoic acid (Sigma-Aldrich, Saint Louis, MO, USA) was used. Recombinant BDNF protein (Peprotech, Rocky Hill, NJ, USA) was treated as positive control and supernatant of BDNF-eMSCs, together or alone with recombinant human TrkB-hyFc (Sino Biological, Beijing, China), were incubated with differentiated SH-SY5Y cells for 1 h at the 37 °C, 5% CO₂ incubator. BDNF-eMSCs were harvested and lysed with RIPA buffer (Thermo Fisher Scientific). Lysate proteins were distinguished using SDS-PAGE, transferred to nitrocellulose membranes (Thermo Fisher Scientific), and incubated with blocking buffer (Thermo Fisher Scientific) for 30 minutes. Immunoblots were performed using primary antibodies against TrkB, PLC-γ, AKT, ERK, Phospho-TrkB (Tyr706/Tyr707), Phospho-PLC-γ (Tyr783), Phospho-AKT (Ser473), Phospho-ERK (Thr202/Tyr204) (Cell Signaling, Danvers, MA, USA), and ACTIN (MP Biomedicals, Santa Ana, CA, USA). Horseradish peroxidase (HRP)-conjugated mouse (GeneTex, Irvine, CA, USA) and rabbit (Cell signaling) secondary antibodies were detected with ECL reagent (Thermo Fisher Scientific) and a ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA).

B35, a rat neuroblastoma cell line, was provided from the American Type Culture (ATCC, CRL-2754). Human neuroblastoma SH-SY5Y cells were obtained from the Korean Cell Line Bank (KCLB 22266, Seoul, Republic of Korea). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum and 1% antimycin A at 37 °C in a humidified atmosphere under 95% air and 5% CO₂.
RAW264.7 cells were procured from the Korean Cell Line Bank (KCLB 40071, Seoul, Republic of Korea) and maintained in RPMI-1640 full medium supplemented with 10% fetal bovine serum and 1% antimycin A. The murine BV2 microglial cell lines were obtained from the KCLB, Seoul, Korea, and were cultured in DMEM containing 10% fetal bovine serum and 1% antimycin A. The cells were maintained in a humid atmosphere of 5% CO₂ at 37 °C.