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NGF is a member of the neurotrophin (NT) family, which also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Neurotrophins are key regulators of development, maintenance, and survival, as well as memory formation and storage. NGF plays a critical role in the stimulation, survival, and phenotypic maintenance of adult basal forebrain cholinergic neurons (BFCNs). It is synthesized in the cortex and hippocampus and retrogradely transported to BFCN cell bodies, triggering processes that lead to the maintenance of normal cellular function and morphology. NGF can upregulate several cholinergic markers—specifically, ChAT activity, gene expression, and protein levels; acetylcholine synthesis and release; and the expression of the vesicular acetylcholine transporter (VAChT).

The biological effects of NGF are closely linked to the specific type of receptor to which it binds. NGF exerts its cellular functions through the action of three distinct types of receptors. It promotes cell survival by binding to the tropomyosin receptor kinase A (TrkA) receptor, yet promotes apoptosis by binding to the p75 neurotrophin receptor (p75 NTR) and sorting nexins. The TrkA receptor belongs to the receptor tyrosine kinase family, a group that also includes TrkB (which binds BDNF and NT-4/5) and TrkC (which preferentially interacts with NT-3). The TrkA receptor is a high-affinity tyrosine kinase receptor classified as a type I transmembrane protein. Ligand-induced activation of TrkA leads to receptor dimerization and the phosphorylation of various residues, which in turn promotes the activation of distinct signaling pathways, including the phospholipase C-γ (PLC-γ), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) pathways.

The HEK293 Human NGF/Canine TRKA Effector Reporter Cell Model—effectively simulates the signal transduction process of NGF/Canine TRKA *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human NGF/Canine TRKA Effector Reporter Cell Model

Figure 2.Dose Response of Recombinant Human β-NGF Protein  in NGF/Canine TRKA Effector Reporter Cell (C19).
 

Figure 3. Inhibition of NGF-induced Reporter Activity by Canine NGF Neutralization Ab in NGF/Canine TRKA Effector Reporter Cell (C19).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  HEK293 Human NGF/Canine TRKA Effector Reporter Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.