β ngf

Cells were grown in medium containing 10% FBS for 24/72 h in the absence or presence of β-NGF (PeproTech, 450-01, Suzhou, China) and/or imatinib (MCE, HY-15463, NJ, USA). The number of living cells was counted on Countless II FL (Life technology) using 0.4% Trypan Blue.

For neurite outgrowth assays, PC-12 cells were seeded in PC-12 differentiation medium at a density of 2.6 × 10⁴ cells per well in 24-well plates, coated with Poly-l-lysine (Sigma-Aldrich). PC-12 differentiation medium consisted of 1% horse serum and 1 ng/mL β-NGF (Peprotech) in RPMI-1640 medium (Harry Perkins Institute of Medical Research). Six hours after seeding, cells were transduced with lentivirus as described above. Cells were incubated with lentivirus overnight and the following day, transduction medium was replaced with PC-12 differentiation medium. Images were acquired four days after cells were initially seeded. Four fields of view were analyzed from each well, with 3 wells analyzed per condition. Images were quantified using ImageJ software (NIH).

Ti foil was purchased from Xinji Metal Materials Co., Ltd., (China). Ammonium fluoride, potassium hydroxide, ethylene glycol, ethanol, and sodium dodecyl sulfate (SDS) were obtained from Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin was obtained from Sigma-Aldrich. Tissue culture plate was purchased from Biosharp. Phalloidin labeled with Alexa Fluor 555, 4′,6-diamidino-2-phenylindole (DAPI), and Trizol reagent were obtained from Invitrogen. A reverse-transcription kit and SYBR Green^® Premix Pro Taq™ HS qPCR Kit II were purchased from Accurate Biotechnology (Hunan) Co., Ltd., and β-NGF, BMP-2, and VEGF were purchased from Peprotech.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of HIV seronegative donors, and monocytes prepared as described above. MMG were generated from isolated monocytes using methods modified from those previously described [34 (link), 37 (link)]. To induce the differentiation of MMG, monocytes were cultured in RPMI-1640 supplemented with 1X GlutaMAX (Life Technologies), 1% penicillin/streptomycin and a mixture of the following human recombinant cytokines: M-CSF (10 ng/mL; Peprotech), GM-CSF (10 ng/mL; Peprotech), NGF-β (10 ng/mL; Peprotech), CCL2 (100 ng/mL; Peprotech), and IL-34 (100 ng/mL; Peprotech) under standard humidified culture conditions (37 °C, 5% CO₂). The monocytes were incubated with 1% serum for the first 3 days, and thereafter, every third day, cells were supplemented with fresh serum-free media containing M-CSF, GM-CSF, NGF-β, IL-34 and CCL2. Cells were cultured for 14 days. Characterization experiments were performed on day 14.

Purified eosinophils were resuspended in RPMI medium (containing 10% and 1% PenStrep) (VWR International, Leuven, Belgium). Eosinophils were stimulated for 4 h at 37 °C and 5% CO₂ with IL-3 (10 ng/mL), IL-13 (50 ng/mL), IL-33 (10 ng/mL), TSLP (10 ng/mL), NGF-β (10 ng/mL), BDNF (50 ng/mL), TNF-α (10 ng/mL), or IL-31 (10 ng/mL) (PeproTech, Cranbury, NJ, USA). Eosinophils were also incubated without any stimulants for 4 h at 37 °C and 40 °C and at pH 5.0, 5.5, 6.0, or 7.0. The pH value of the medium was adjusted with HCl (Sigma-Aldrich, St. Louis, MO, USA) and NaOH (Carl Roth, Karlsruhe, Germany) and determined with a pH electrode.