Assessing the activity of caspase-activated pannexin-1 channels by flow cytometry

Pannexin 1 (PANX1) is a ubiquitously expressed plasma membrane channel that plays an important role in maintaining cellular homeostasis through the release of small signalling metabolites. Under cell death settings, PANX1 channels can become activated by caspase-3/7 cleavage. This leads to the release of chemotactic 'find-me' signals and anti-inflammatory 'good-bye' signals. Additionally, caspase-activated PANX1 channels can regulate the fragmentation of dying cells. Due to the various cellular pathways that PANX1 channels are involved in, this membrane channel has emerged as an attractive target for novel therapeutics. To assess the effectiveness of pharmacological inhibition on caspase-activated PANX1 channels, the cell-impermeable nucleic acid-binding dye, TO-PRO-3 can be used due to its ability to be selectively taken up into apoptotic cells through caspase-activated PANX1 channels. Here we describe a robust flow cytometry-based protocol utilising Annexin A5 and TO-PRO-3 staining to accurately monitor apoptotic cell death and PANX1 channel activity. This approach can be easily adapted to investigate pharmacological compounds that have the potential to inhibit caspase-activated PANX1 channels.