Effects of low calcium Locke's buffer on neuronal viability. Neurons were exposed to MEM (controls), Locke's buffer with and without Ca2+ and/or 1 mM EGTA for 15 min or to a low calcium Locke's buffer (LCLB) containing 920 μM Ca2+ and 1 mM EGTA for 2.5, 5, 7.5, 10, 12.5 and 15 min. Viability was assessed using the colorimetric MTT assay, where a loss of viability is reflected in a decrease in absorbance at λ = 540 (A540). The substitution of normal Locke's buffer for one containing no added calcium results in a loss of neuronal viability (27%, Welch's test, P < 0.005, n = 8, indicated by a superscript a). This solution was not used for the mefloquine studies because control experiments showed that glutamate elicited a calcium response in neurons exposed to this buffer, presumably because the rinsing procedure did not remove all residual free calcium. A 10 min exposure to LCLB was required to reduce viability below that of the calcium-free Locke's buffer (one way ANOVA and Dunnett's test, P < 0.05, n = 44, * designates a significant change). Use of this buffer does not elicit a glutamate response, and therefore should not contain substantial amounts of free calcium. Experiments that utilized this buffer to investigate the effect of mefloquine on neuronal calcium homeostasis were performed within this ten-minute window.