Fig. 2

TNFα synergizes with IFNγ causing reduced OL survival and inhibiting differentiation. Expanded OPCs were plated in DMEM differentiation media for 24 h and then treated with various combinations cytokines for 48 h. A Differentiating OLs were treated with increasing concentrations of TNFα alone (gray bars) or increasing concentrations of TNFα + 100 U/ml INFγ (black bars) and alamarBlue® (AB) fluorescence was quantified to determine cell viability. B Cells were treated as in A with increasing concentrations of TNFα alone (black bars) or TNFα + 5, 10, or 20 U/ml INFγ (light gray, dark gray, or white bars respectively) and AB fluorescence was quantified to determine cell viability. C Image quantification of anti-MBP immunostaining of the same cultures as in B was used to measure OL differentiation. D Comparison of MBP expression in untreated (No insult) or 10 U/ml INFγ + 1 ng/ml TNFα treated differentiating OLs. E Viability and OL differentiation raw values of no insult versus 10 U/ml INFγ + 1 ng/ml TNFα demonstrate the reproducibility of the combined cytokine toxicity. Coefficient of variation (CV) values for cell viability were 7.47 and 6.14 % and for OL differentiation were 11.37 and 19.16 % for No insult or TNFα+INFγ, respectively. CV values <20 % were considered in the acceptable range