1 1 dimethyl 4 diphenylacetoxypiperidinium iodide 4 damp

The water-soluble tetrazolium-1 (WST-1) assay kit (Roche Diagnostics GmbH, Germany) was used to determine the viability of LIM-2405 and HT-29 cells. WST-1 is cleaved to form formazan dye via a complex cellular interaction at the cell surface. This interaction is contingent on the viable cells’ production of glycolytic nicotinamide adenine dinucleotide phosphate (NADPH). Hence, the amount of formed formazan dye correlates with the number of viable cells in the culture. LIM-2405 and HT-29 cells were seeded and cultured at 1 × 10⁴ cells per well in 96-well plates for 24 h (hrs). Cells were then treated with various concentrations of the general muscarinic receptor blocker, atropine (Sigma-Aldrich, Australia) for 1–48 h, selective M3R blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) (Abcam, Australia), cholinergic agonist, carbachol (Abcam, Australia) and acetylcholinesterase inhibitor, donepezil (Abcam, Australia) for 8 h. All treatments were performed in triplicates, and three independent experiments were conducted. WST-1 reagent (10 µL) was added to each well and incubated at 37 °C for 1 h. Cellular proliferation at the absorbance of 450 nm was measured using a microplate reader (Varioskan Flash, Thermo Scientific, Australia).

The water-soluble tetrazolium-1 (WST-1) assay kit (Roche Diagnostics GmbH, Penzberg, Germany) was used to determine the viability of CT-26 cells. WST-1 is cleaved to form formazan dye via a complex cellular interaction at the cell surface. This interaction is contingent on the viable cells’ production of glycolytic nicotinamide adenine dinucleotide phosphate (NADPH). Hence, the formed formazan dye correlates to the number of viable cells in the culture. CT-26 cells were seeded and cultured at 1 × 10⁴ cells per well in 96 well plates for 24 h. Cells were then treated with various concentrations of the general muscarinic receptor blocker, atropine (Sigma-Aldrich, Australia), selective M3R blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) (Abcam, Adelaide, SA, Australia), cholinergic agonist, carbachol (Abcam, Australia) and acetylcholinesterase inhibitor, donepezil (Abcam, Australia) for 8 h. All treatments were performed in triplicates, and three independent experiments were conducted. WST-1 reagent (10 µL) was added to each well and incubated at 37 °C for 1 h. Cellular proliferation at the absorbance of 450 nm was measured using a microplate reader (Varioskan Flash, Thermo Fisher Scientific, Scoresby, VIC, Australia).