TMRE-mitochondrial membrane potential assay.

A mitochondrial membrane potential assay kit (Abcam) was used according to the supplier's instructions to quantify modifications in mitochondrial membrane potential. The assay uses TMRE (tetramethylrhodamine) dye to label healthy mitochondria in living cells. TMRE reagent labels active mitochondria but fails to accumulate with depolarized, damaged mitochondria that have a decreased mitochondrial membrane potential. We used this assay in combination with the LIVE/DEAD fixable aqua dead cell stain to determine cell viability. HPNs were treated with or without HIV recombinant proteins for 24 h and stained with TMRE for 15 min, followed by aqua DEAD cell stain for 30 min. CCCP was used as a positive control. Live cells were collected and analyzed by flow cytometry.