Cell Proliferation Kit II (XTT)

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	- 49 μL of XTT and 1 μL of electron‐coupling reagent diluted 1 : 1 with SFM was added to each well and the plate incubated at 37°C for 4 h.

Protocol tips
- 49 μL of XTT and 1 μL of electron‐coupling reagent diluted 1 : 1 with SFM was added to each well and the plate incubated at 37°C for 4 h.

Publication protocol

Cell viability, cytotoxicity, and proliferation were assessed using 2,3‐Bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide (XTT, Cell proliferation kit II), Lactate dehydrogenase (LDH, CytoTox 96 assay kit) and 5‐bromo‐2‐deoxyuridine (BrdU, Cell proliferation ELISA colorimetric kit) incorporation, respectively. The assay was formatted by seeding SH‐SY5Y cells in laminin‐coated 96‐well plates at a density of 5 × 104 cells/well for 24 h. The cells were serum starved for 12 h in serum free medium (SFM) and were pre‐treated for 4 h with different concentrations of Liraglutide. Medium was removed and the cells were exposed to various concentrations of MG for 12 h. Supernatant was removed (to be used for LDH assay) and 49 μL of XTT and 1 μL of electron‐coupling reagent diluted 1 : 1 with SFM was added to each well and the plate incubated at 37°C for 4 h. XTT measures cell viability based on the activity of mitochondrial enzymes in a live cell that reduces XTT to a highly water‐soluble product, λmax 492 nm, which is directly proportional to the number of living cells in the sample. The plate was gently shaken for 5 min on Microtitre plate shaker (Stuart, Staffordshire, UK) and absorbance measured at 492 nm and 690 nm in a FLUOstar Omega microplate reader (BMG LABTECH, Aylesbury, Bucks, UK).

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