Rat Patrice, Olivier Elodie, Tanter Caroline, Wakx Anaïs, Dutot Mélody
UMR 8638 CNRS COMETE, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, 4 avenue de l'Observatoire, 75006 Paris, France.
Soliance-Givaudan, Route de Bazancourt, 51110 Pomacle, France.
J Biol Methods. 2017 Jan 20;4(1):e64. doi: 10.14440/jbm.2017.136. eCollection 2017.
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.
YO-PRO-1检测可对P2X7受体激活进行定量评估。P2X7受体与多种病理状况相关，包括感染性、炎症性、神经性、肌肉骨骼疾病、疼痛和癌症。由于P2X7受体分布广泛，来自不同来源的大多数原代细胞和细胞系均可使用。为了研究化学物质或生物制剂对P2X7受体的激活作用，我们建立了一种基于微孔板的细胞计数方法，以准确快速地定量P2X7受体的激活情况，该激活会导致细胞膜上形成大孔。YO-PRO-1检测基于细胞在通过形成孔激活P2X7受体后摄取并结合YO-PRO-1染料至DNA的能力。将细胞接种于96孔板中，并与受试化合物孵育适当时间。然后将微孔板与YO-PRO-1染色溶液孵育10分钟。在10分钟染色时间后，使用微孔板读数器在1分钟内读取荧光信号。该方法比流式细胞术更简便，所需操作步骤更少。基于96孔板的YO-PRO-1检测是一种可重复且快速的方法，可用于研究亚坏死浓度下有毒物质对P2X7受体的激活作用以及拮抗剂对P2X7受体的抑制作用。
