Fig. 5

OL-HSPB8-EVs do not promote cell death in C20 cell line and primary mixed neural cultures. A–F Flow cytometry quantification of early apoptosis by using the JC1 dye to detect the mitochondrial membrane potential (A–D) and by using Annexin V to measure phosphatidylserine exposure E, F in the human microglial C20 cell line and a primary mixed neural culture stimulated with OL-EVs or OL-HSPB8-EVs. Cells were either activated with TNFα/IL1β or non-activated. The positive control was stimulated with PMA or staurosporine dependent for each experiment. Data are presented as mean percentage ± SEM (n = 3 biological replicates per group). Graphpad was used to determine significance by using a one-way ANOVA statistical analysis (*P < 0.05, **P < 0.01). G Relative qPCR expression of the apoptosis-related genes Caspase1, Caspase8, FAS, IL1β, TRAIL, FADD, and VEGF in human C20 cells stimulated with OL-EVs or OL-HSPB8-EVs either non-activated, or TNFα/IL1β activated. As a positive control, the apoptosis inducer staurosporine was used. Values are represented as the mean ± SEM of three independent biological replicates. Data were normalized to non-stimulated equals 1. One-way ANOVA was used to determine statistical significance were P < 0.05 was considered as statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)