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

Patrice Rat,Mélody Dutot,Elodie Olivier,Anaïs Wakx,Caroline Tanter

doi:10.14440/jbm.2017.136

Abstract

The 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.

Highlights

High throughput screening methods are emerging approaches in toxicology, allowing a quick and efficient toxicity evaluation through a battery of assays that target cellular processes [1]
Retinal cells were incubated with amyloid β peptide or oxysterols, involved in degenerative diseases such as Alzheimer and age-related macular degeneration, the YO-PRO-1 assay that we describe here was used to study P2X7 receptor activation
The first studies looking at P2X7 activation through YO-PRO-1 uptake used fluorescence microplate readers with filter detection systems; we were among the first to use a monochromator-based system [35,36,37,38,39,40,41,42,43]

Summary

Introduction

High throughput screening methods are emerging approaches in toxicology, allowing a quick and efficient toxicity evaluation through a battery of assays that target cellular processes [1]. A fast and reproducible cell- and 96-well plate-based method for the evaluation of P2X7 receptor activation using YO-PRO-1 fluorescent dye. The first studies looking at P2X7 activation through YO-PRO-1 uptake used fluorescence microplate readers with filter detection systems; we were among the first to use a monochromator-based system [35,36,37,38,39,40,41,42,43].

Results

Conclusion