Ngf β

Second passage hUCMSCs were seeded in DMEM/F12 containing 10% fetal bovine serum. After the cells reached 90% confluence, the medium was replaced with DMEM/F12 medium containing 1% fetal bovine serum, followed by culture for an additional 48 hours at 37°C in a humidified 5% CO₂ incubator. The hUCMSC-conditioned medium was collected, filtered through a 0.22-μm filter, and immediately frozen at −80°C until use. DMEM/F12 medium containing 1% fetal bovine serum was used as a control. The conditioned media were analyzed using human brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), nerve growth factor (NGF)-β and vascular endothelial growth factor (VEGF) ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocols. All samples were analyzed in triplicate, and the optical density was measured at 450 nm (Wellscan MK3, Labsystems Dragon, Helsinki, Finland).

Mice were anesthetized with a lethal exposure to isoflurane and intracardially perfused with ice cold 0.9% saline prior to the dissection and collection of hindpaw skin.
Hindpaw hairy skin was homogenized in ice-cold RIPA buffer/protease inhibitor cocktail and centrifuged for 20 min (4 °C; 18,000 rcf). Each sample’s total protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific; Waltham, MA). ELISAs for CGRP (MyBioSource; San Diego, CA), substance P (SP; Enzo; Farmingdale, NY), and nerve growth factor β (NGFβ; Boster Bio; Pleasanton, CA) were run according to manufacturer’s instructions. All samples were run in duplicate and absorbance ratios were read at 450 nm. Protein concentration was determined by comparison to linearized protein concentration standards, and analyses were conducted using the mean concentration for the duplicate wells for each sample.