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. 2015 Jun 9;9:203. doi: 10.3389/fncel.2015.00203

FIGURE 6.

FIGURE 6

Nav channel blockers, calcium chelator, mitochondria protectors, and antioxidants prevent DCF fluorescence induced by ACM-hSOD1G93A. (A) Flow diagram of experiment. Spinal cultures (4 DIV) were exposed for 30 min to ACM-hSOD1G93A alone or together with Nav channel blockers: mexiletine (25 nM), spermidine (10 μM), or riluzole (100 nM); calcium extracellular chelator EGTA (200 μM); mitochondria protectors: CsA (10 μM) or Ru360 (5 μM); or the antioxidant Trolox (1 μM). Next, cultures were incubated with the membrane permeable ROS/RNS probe CM-H2DCF-DA and DCF fluorescence was measured 30 min later in neurons using a combination of real-time fluorescence and phase-contrast imaging. (B–E) Graphs showing the intensity (arbitrary unit fluorescence; AUF) of DCF fluorescent cells after being treated with ACM-hSOD1G93A alone or with the diverse Nav channel blockers (B), calcium chelator EGTA (C), mitochondria protectors (D), or antioxidant Trolox (E). Values represent mean ± SEM from at least three independent experiments performed in duplicate, analyzed by One-Way ANOVA followed by a Tukey post hoc test. ∗∗p < 0.01, ∗∗∗p < 0.001 relative to control conditions, and ##p < 0.01, ###p < 0.001 compared to DCF fluorescence with ALS-causing ACM to at 4 DIV without blockers.