4.4. Flow Cytometry Analysis

Flow cytometry was used to measure mitochondrial mass in EHV-1 infected neurons with MitoTracker Green FM (490/516 nm [Em/Ex]). Neurons (10⁶ cells/mL) at 2, 24, and 48 h p.i. were stained with MitoTracker Green FM (200 nM; Thermo Fisher Scientific, Waltham, MA, USA) for 10 min in 37 °C, according to the manufacturer’s protocols. It is a non-fluorescent dye in aqueous solutions, but becomes fluorescent once it accumulates in the lipid environment of mitochondria, regardless of membrane potential. Samples were analyzed by BD LSR Fortessa cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Non-infected neurons stained with MitoTracker Green FM were used as a positive control and non-infected neurons unstained with MitoTracer Green FM were used as a negative control.