2.19. Annexin V phosphatidylserine (PS) apoptosis assay

In order to determine early apoptosis, phosphatidylserine (PS) exposure was measured using the PS‐binding protein Annexin V conjugated to a fluorescent FITC label. Therefore, 2.5 × 10⁵ primary microglia cells or 5 × 10⁴ BV2 and C20 cells were seeded in complete medium. The next day, complete medium was replaced by reduced medium and cells were stimulated with 1 × 10⁹ nEVs or aEVs per 1 ml reduced medium for 24 h, followed by TNFα‐activation for 24 h. Apoptosis was induced using 1 μM Valinomycin (Enzo, BML‐KC140) stimulation for 24 h. Cells were harvested, centrifuged and resuspended in 100 μl of assay buffer (Invitrogen, PNN1001). Per reaction, 5 μl Annexin V‐FITC (Invitrogen, 11‐8005‐74) and 2.5 μl 7AAD dye (BD Pharmingen, 51–68981E) were incubated in the dark for 15 min at room temperature. Afterward, cells were centrifuged, washed and resuspended in 1× assay buffer. Single cell fluorescent intensities were quantified by flow cytometry on a BD‐LSRFortessa flow cytometer (BD Biosciences) using FACSDiva software (BD Biosciences).