Figure 3.

Small-molecular-weight nucleic acid-binding dyes stain pyroptotic BMMs with differential kinetics according to their size. (a) Fluorescent intensities over time of Sytox Blue, PI, and EtBr₂ in non-fluorescent wild-type BMMs stimulated with RodTox in the absence of supplemental glycine. PI and EtBr₂ staining is significantly delayed relative to Sytox Blue, P=0.022 and P=0.040, respectively, rANOVA. EtBr₂ staining is significantly delayed relative to PI staining, P=2.9E-12, rANOVA. (b) Fluorescent intensities over time of Sytox Blue, PI, and EtBr₂ in non-fluorescent wild-type BMMs stimulated with RodTox in the presence of 5 mM glycine. PI and EtBr₂ staining is significantly delayed relative to Sytox Blue, P=0.00011 and P=2.94E-16, respectively, rANOVA. EtBr₂ staining is significantly delayed relative to PI staining, P=7.76E-9, rANOVA. The delay in PI and EtBr₂ staining relative to Sytox Blue staining is not significantly distinct±5 mM glycine, P=0.097 and P=0.394, respectively, rANOVA. In both a and b, Sytox Blue was paired with either PI or EtBr₂ in separate imaging wells run in parallel and imaging data were aligned setting influx of Sytox blue to time 0 : 00 (see ‘Materials and Methods’). Lines represent the population mean and the shaded areas the 95% confidence interval. Data in a and b are pooled from two independent experiments and ‘n’ indicates total number of individual BMMs analyzed. ‘−Gly’, no supplemental glycine; ‘+Gly’, 5 mM supplemental glycine.