L glutamine

Normal human mammary cells MCF10A and human breast cancer cell lines, MCF7, T47D and BT-474 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MCF10A were cultured in special medium (Procell, China). MCF7 and T47D cells were maintained in DMEM high glucose medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, USA). BT-474 cell were maintained in RPMI-1640 (Gibco, USA) supplemented with 20% fetal bovine serum (Gibco, USA), 10 μg/mL Insulin (Beyotime, China) and 2mM L-glutamine (Procell, China). Cells were incubated at 37 °C in an atmosphere of 5% CO2.

Human small airway epithelial cells (SAECs) were purchased from Procell Life Science & Technology Company (Wuhan, China) and cultured in DMEM high glucose supplemented with 10% FBS, 1% penicillin–streptomycin solution, and 1% l-glutamine (all purchased from Procell, Wuhan, China) at 37 °C with 5% CO₂. SAECs were seeded in 24-well plates to 90% confluency, washed once with PBS, and 20 μg/mL NETs or DMEM were added. The total volume in each well was kept equal by adding DMEM medium. After 24 h treatment, SAECs were harvested for RNA sequencing.

Neural stem cells were obtained from the spinal cord tissues of day 14 embryonic SD rats, which were isolated and cultured per established protocol (Lei et al., 2009 (link)). Briefly, spinal cord tissues were separated and cut into small pieces, and then the pieces were digested with trypsin to collect cell suspension. After centrifuging at 1,500 rpm for 6 min, the supernatant was discarded. The acquired NSCs were plated on cell culture flasks with a complete medium (Procell, Wuhan, China) containing Dulbecco’s Modified Eagle Medium/F-12, 20 ng/mL epidermal growth factor, 20ng/mL basic fibroblast growth factor, 2% B-27 supplement, 1% Penicillin-Streptomycin and 4mmol/L L-glutamine. The NSCs were cultured in a chamber with 5% CO₂ at 37°C and passaged 1–2 times 1 week. The lentiviral vectors overexpressing NGF (pLVX-IRES-ZsGreen-rat NGF) were synthesized by Biobuffer Biotech Service (Wuhan, China). They were used to transfect the NSCs at 3rd passage and exponential growth period.
For the differentiation in vitro, NGF-NSCs were seeded in the mediums composed of DMEM/F12 (Procell), 1% N2 supplement (Thermo Fisher Scientific, Waltham, WI, United States), 2mmol/L L-glutamine (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Aladdin, Shanghai, China) and 1% Fetal Bovine Serum (Excell Bio, Shanghai, China) for 7 days.