Ts2 fl

The NPCs were isolated using the method as previously described [4 (link)]. First, the collected NP tissues were cut with a size of 1 mm³ and incubated with trypsin (0.25%, PB180228, Procell, Wuhan, China) for 30 min, followed by centrifugation at 1,000g (E2658, Beyotime, Shanghai, China) for 10 min and incubation with collagenase type II (40508ES60, Qcbio Science & Technologies Co., Ltd, Shanghai, China) at 37°C for 4 h. Later, the treated NP tissues were filtered with a 200-mesh filter (S4203, Aladdin, Shanghai, China), maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F medium (with the inclusion of 1% penicillin–streptomycin mixture solution, PM150312A, Procell, Wuhan, China) supplemented with 20% of fetal bovine serum (164210, Procell, Wuhan, China) and cultured in an incubator (BC-J80, BoXun, Shanghai, China) at 37°C with 5% CO₂. The cell medium was replaced 2–3 times a week.
When the NPCs grew attached, the morphology of primary NPCs was observed (magnification 200×) under an inverted microscope (Ts2-FL, Nikon, Tokyo, Japan) as appropriate. NPCs with passage ≤3 (P ≤ 3) were used for the subsequent experiments in the present study [22 (link)–24 (link)].

DGF cells were passaged into a confocal dish at a density of 3.0 × 10⁵ cells/mL. After being cultured for 20 h at 28 °C, DGF cells were transfected with pAcGFP1 vector (Takara, Kusatsu-Shi, Chuo-Ku, Japan) using Lipofectamine 3000 reagent and pAcGFP1 vector. A total of 5 µg of pAcGFP1 vector was diluted by 250 µL of Opti-MEM medium (Gibco, Grand Island, NY, USA). The diluted pAcGFP1 vector was mixed with Lipofectamine 3000 and incubated at RT for 15 min. This mixture was then added to a confocal dish. After 48 h, green fluorescence signals were detected using a Ts2 FL (Nikon, Minato-Ku, Kadoma-Shi, Japan).

The Passage 4/5 ASCs kept in complete medium was replaced with serum-free DMEM medium with 20 ng/ml of bFGF (known neuronal inducer) and 5% PAN serum replacement. The cells were maintained for 7 days in the same medium with periodical change of fresh medium for every 3 days. Later the bFGF containing medium was removed and washed with DPBS. Serum-free medium with 5% PAN serum replacement (Panexin NTA, PAN Biotech catalogue no. PO4-95700) was added to the cells. This temporal exposure (mimics in vivo exposure) of bFGF aids neuronal induction. The morphological changes were observed under Nikon Ts2FL phase contrast microscopy [22 (link)].