A fast and reproducible cell- and 96-well plate-based method for the evaluation of P2X7 receptor activation using YO-PRO-1 fluorescent dye

Abstract

A B S T R A C TThe YO-PRO-1 assay provides a quantitative estimation of P2X7 receptor activation. P2X7 receptor is associated to pathological conditions including infectious, inflammatory, neurological, musculoskeletal disorders, pain and cancer. Most primary cells and cell lines from diverse origin may be used thanks to the ubiquitous distribution of P2X7 receptor. To study the activation of P2X7 receptor by chemicals or biological agents, we established a microplate-based cytometry protocol to accurately and rapidly quantify the activation of P2X7 receptor that leads to the formation of large pores in cell membranes. The YO-PRO-1 assay is based on the ability of cells to incorporate and bind YO-PRO-1 dye to DNA after activation of P2X7 receptor through pore formation. Cells are seeded in 96-well plates and incubated with the compound being tested for the appropriate time. The microplate is then incubated for 10 min with YO-PRO-1 staining solution. After the 10 min staining time, fluorescence signal is read using a microplate reader in 1 min. This procedure is easier and requires less handling steps than flow cytometry. 96-well plate based YO-PRO-1 assay is a reproducible and fast method to study both P2X7 receptor activation by toxic agents at subnecrotic concentrations and P2X7 receptor inhibition by antagonists.

Detection of active cell death markers in rehydrated lichen thalli and the involvement of nitrogen monoxide (NO).

“…On the other hand, YO-PRO-1 enters only those cells whose membrane permeability is increased due to opening of P2X7 small cation channels caused by ATP extracellular release. This route is widely used in animal cells for apoptosis detection (Virginio et al 1999;Fujisawa et al 2014; Fluorescence was quantified in a plate reader at 1 h intervals for 24 h: a) green channel (λ exc 360 -λ em 460 nm) and b) blue channel (λ exc 485λ em 528 nm) Rat et al 2017). Green algae have been reported to have P2X receptor homologs and they have also been found in three basal fungi Allomyces macrogynus, Spizellomyces punctatus, and Batrachochytrium dendrobatidis (Cai 2012).…”

“…Taking advantage of changes in plasma membrane permeability and chromatin condensation in apoptotic cells, DNA fluorescent markers such as YO-PRO-1, TOPRO, SYTO 13 and Hoechst 33342 can be applied. The fluorescence emitted by YO-PRO-1 has been used in multiple animal cell studies since this molecule enters selectively apoptotic cells after the opening of P2X7 small cation channels upon ATP extracellular release (Virginio et al 1999;Fujisawa et al 2014;Rat et al 2017). Caspase and caspase-like activities assessment by means of specific fluorescent substrates has been extensively used for PCD study.…”

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

“…Methods to study the receptor have evolved and been adapted over the years to facilitate this research 2,3,4,5 . Here, we describe a live-cell flow cytometry method to investigate the multiple functions of P2X7 receptors in adult neural progenitor cells derived from the subventricular zone (SVZ) and the hippocampal dentate gyrus.…”

“…This leads to cytoskeletal rearrangement, transmembrane pore formation, and, potentially, apoptosis and/or necrosis 7 . Traditionally, this function of P2X7 is quantified by the uptake of large molecular weight dyes such as YO-PRO-1 or ethidium bromide, which fluoresce when intercalated with DNA 3,8 . Plate reader methods, which are rapid and allow for upscaling, generally do not allow for the observation of kinetics.…”

The Role of the P2X7 Receptor in Ocular Stresses: A Potential Therapeutic Target

“…P2X7 receptors show complex gating behavior: several seconds of ATP exposure induces P2X7 receptors’ dilatation from a channel that allows for the passage of small cations to a pore that allows for permeation of larger cations and dyes such as YO-PRO-1 (see YO-PRO-1 staining protocol in [25]). P2X7 receptor activation triggers numerous cellular effects from oxidative stress to apoptosis, including inflammation.…”