2.3. Crystal Violet Cell Proliferation Assays

After plating, cells were left to adhere overnight, before stimulation with a range of concentrations (0 to 10 mM) of the ammonia donor, NH₄Cl, in the absence or presence of 100 nM CNP, in DMEM supplemented with 1% (v/v) FCS and 1% (v/v) antimycotic/antimicrobial, to reduce the basal rate of proliferation. At the indicated time points (24, 48 and 72 h), cells were washed twice with PBS, before being fixed with 4% (w/v) paraformaldehyde-containing PBS and stored at 4 °C prior to staining with 0.075% (w/v) crystal violet solution for 15 min, followed by subsequent washing in water and drying overnight. The stained cells were dissolved in 10% (v/v) acetic acid and left for 30 min, before measuring the optical density at 595 nm using a Mithras LB940 Multimode Plate reader (Berthold, Harpenden, UK). Experiments using GPNT cells also included treatments in the absence and presence of 1 mM SNP.