Thus, transport of chloride into liposomes produces a smaller response: after lysis of the liposomes, the final level of normalized fluorescence intensity (F/F0) is higher by 0.15-0.20 in bicarbonate solution than in nitrate solution. Secondly, bicarbonate solutions are not stable over time unless stored under a CO2 atmosphere, thus these should be prepared freshly. The pH value of freshly prepared 225 mM NaHCO3 solution is around 8.3, and therefore a small amount of H2SO4 is added, to decrease the pH to 7.5-7.8, to better reflect physiological conditions. However, release of CO2 over time leads to a decrease of the bicarbonate concentration and a basification of the solution, a process that is faster at lower pH. Due to quenching of lucigenin fluorescence by bicarbonate, using the same NaHCO3 solution over multiple days/weeks, results in different values of initial and final fluorescence levels. Therefore, the best practice is to prepare a fresh bicarbonate solution daily, implying the preparation of a fresh lucigenin solution for lipid film hydration as well.