Jinma analysis of nerve growth factor detection method - Database & Sql Blog Articles

1. PC12 Cell Culture:

PC12 cells were cultured in a CO₂ incubator with 7.5% CO₂ and saturated humidity at 36°C. The culture medium was replaced by 2/3 every two days. When the cells reached near confluence, they were treated with culture medium containing 1 mmol/L EGTA for at least 6 hours. After washing three times with RPMI-1640/water (1:1), the cells were detached and reseeded in 1:3 or 1:4 flasks. The doubling time of PC12 cells is approximately 2.5 to 4 days.

2. NGF Activity Detection:

1) Cells were suspended at a concentration of 10⁶ cells/ml using the same culture medium. Then, 0.5 ml of the cell suspension was mixed with serially diluted NGF samples or standards in each 35 mm dish. A control dish without NGF was also prepared. The dishes were incubated for 20–24 hours in a CO₂ incubator at 37°C with 7.5% CO₂.

2) After incubation, the cells were observed under a phase-contrast microscope. Cell clusters were counted, and those with fibrillar extensions were considered positive. A dose-response curve was plotted based on the dilution series, allowing the quantification of NGF in the sample.

NGF exerts its biological effects by binding to its receptor, internalizing through receptor-mediated endocytosis, forming vesicles that travel along axons, and transporting the molecule retrogradely toward the cell body. This process activates signaling pathways such as tyrosine kinase, calcium signaling, and cyclic adenosine, which regulate the expression of specific structural or functional genes in target cells, ultimately influencing their function and survival.