2.7. Flow Cytometry

Apoptosis and necrosis were detected by differential staining with annexin V (early and late apoptotic cells) and propidium iodide (PI) (necrotic cells only) using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and PI (#V13241; ThermoFisher Scientific), according to manufacturer’s instructions. Briefly, BV2 cells were treated with 1000 µM of AZE for 24 h. Cells were washed in cold PBS, re-centrifuged and 1 × 10⁶ cells were suspended in 1 × annexin-binding buffer. Cells were stained with Alexa Fluor 488 Annexin-V and propidium iodide (PI) for 15 min then analysed by flow cytometry. Unstained and single stained controls were used for compensation to correct for fluorescence photobleaching and gating (Supplementary Figure S1). FloJo software was used to analyse and curate flow cytometry data.