exMito activates APCs in vitro. T-cell–depleted BALB/c splenocytes were treated with 4 μg or 12 μg of mitochondria for 24 hours; cells were stained with (A) CD86 and (B) MHC-II antibodies, respectively. A representative histogram and the calculated MFI analyzed by flow cytometry is shown (n = 3). (C) Changes in cytokine levels in cell culture supernatants between control and 4 μg of mitochondria-treated splenocytes is shown in volcano plot. Fold change relative to control demonstrated on x-axis and inverse log P value demonstrated on y-axis. Chemokines and cytokines highlighted in red were noted to be significantly different (n = 6). (D-E) The impact of exMito on different APC subpopulations including B cell, macrophages/monocytes, and dendritic cells were further characterized (gating protocol described in supplemental Figure 8B). Expression of (D) CD86 and (E) MHC-II quantified as fold change in MFI is shown (n = 4-8). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001; n, number of independent experiments.