Figure 7.

MESM exposure during the assay does not affect viability as measured by the CTB assay of membrane integrity as measured by CytoTox Green assay. Following Mn treatment and ionophore extraction/treatment, the ionophore/PBS mixture was removed from Q7 cells (A) or HEK cells (B) and then put back into their regular DMEM for 24 h at 37 °C. An injection of 20 μl of CTB reagent was added to each well and returned to the incubator for another 2 h before measuring the absorbance of each well at 590 nm. n = 3–6 biological replicates for Q7 cells (A) and n = 5 for HEK cells (B). The CTB assay relies on viable cells to reduce the reagent resazurin to a fluorescent product, see under “Experimental procedures.” Signals of vehicle conditions were normalized to 100%, and a two-way ANOVA with Sidak's multiple comparisons test to vehicle for every condition was used. Significance from vehicle is denoted by the following: ***, p < 0.001; ****, p < 0.0001. C, separate measure of toxicity was used as validation at the most effective extraction and the least toxic by CTB conditions (3 μm compound) in Q7 cells called CytoTox Green. It is based on membrane integrity, so that a dye will fluoresce if it binds DNA; see under “Experimental procedures.” n = 3 of independent biological replicates for all. Error bars shown represent standard deviation of the average across biological replicates. One-way ANOVA with Dunnett's multiple comparisons only shows statistically significant difference from vehicle in the positive control, which was 0.1% Triton X-100.