Acetylcholine iodide (Acetylcolina) is a neurotransmitter found at neuromuscular junctions, autonomic ganglia, parasympathetic effector junctions, a subset of sympathetic effector junctions, and at many sites in the central nervous system.

Acetylcholine iodide (Ach) stimulated SBC3 cell proliferation, adhesion and migration toward fibronectin (Fn) in a dose-dependent manner[1]. ACh ameliorates TNF-α-induced calpain activation by decreasing p38-MAPK phosphorylation and enhancing calpastatin expression. It elicits an anti-apoptotic effect through the activation of the muscarinic ACh receptor (MAChR) and the activation of anti-oxidant systems. ACh increases cell viability and decreases TNF-α-induced apoptosis in H9c2 cells[2].

Acetylcholine can inhibit pro-inflammatory cytokine release and protect against cardiomyocyte injury[2].

The SBC3 cells are seeded at 2000 cells/well in treated 96-well plates maintained with RPMI1640 medium containing 10% (v/v) FCS for 24 h. The medium is removed and replaced with 100 μl RPMI1640 containing 1% (v/v) ITS. The cells are incubated in serum free medium for 24 h to synchronize the cell cycle. Then another 100 μl serum-free medium containing the agonist/antagonist under investigation is added. The antagonist is added 30 min before Ach(Acetylcholine iodide). At this time, the cells in a duplicate plate are given 100 μl serum-free medium and a MTT assay is used to determine live cell numbers before treatment with the agonist/antagonist as measured by the absorbance (OD) at 550 nm/650 nm. After 24, 48 or 72 h culture, the remaining cells are assayed to yield post-agonist/antagonist cell numbers using the same method. All the experiments are performed in triplicate.(Only for Reference)