PEVs interact with BM MKs in vitro and in vivo. Human platelets were labeled with either PKH67 (membrane marker), CMPTX (cytosol stain), or MitoTracker Red (mitochondrial marker) and activated with thrombin (0.1U/mL) and collagen (1 μg/mL) to generate PEVs. (A) PKH67-labeled PEVs were incubated with murine BM-derived MKs and fixed over a time course of 30 minutes, 1 hour, and 24 hours. Cells were then harvested and stained with Hoechst (H33342) before confocal microscopy analysis. Representative images are z-stack maximum projections. (B) CMPTX-labeled and (C) MitoTracker–labeled PEVs were incubated with isolated, cultured MKs for 24 hours. Cells were then fixed, permeabilized, stained with Hoechst, and examined by confocal microscopy. The images were taken at different confocal planes with 5 µm z-stack steps. The numbers represent a specific slice (z stack) number/total number of slices. (D) Wild-type (MPL⁺), CMPTX-labeled platelets were infused into cMpl^−/− mice before an LPS injection, as in Figure 1. Mice were euthanized after 20 hours, BM was flushed, incubated with anti-CD41 Ab, and sorted for CMPTX⁺/CD41⁺ BM cells by flow cytometry. Sorted populations were fixed, stained with Hoechst, and imaged by confocal microscopy. (E) Wild-type (MPL⁺), CMPTX-labeled PEVs were cultured with freshly isolated cMpl^−/− BM over 72 hours. Cultures were then stained with anti-CD41 and sorted for CMPTX⁺/CD41⁺ cells. Sorted populations were fixed, stained with Hoechst, and imaged by confocal microscopy. Scale bars, 10 µm.