RESUMO
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.
RESUMO
PURPOSE: We evaluated (1) 4 multipurpose lens care solutions and 3 contact lenses (soft and rigid) for cytotoxicity according to ISO 10993-5 standard (medical device biocompatibility) and (2) the protective effects of a marine cationic solution and hyaluronic acid. METHODS: Low water soft lens, high water soft lens, and rigid lens were laid on a conjunctival cell line after being soaked in multipurpose solution (Optifree Express, Renu, Solocare Aqua, or Menicare Plus). Cell morphology was microscopically observed, and cell viability was evaluated using the neutral red test. Apoptosis was assessed after direct contact of multipurpose solutions (MPS) with conjunctival cells using fluorescence microscopy and flow cytometry. The ability of a controlled ionization marine solution and hyaluronic acid to prevent multipurpose solution's cytotoxicity was finally evaluated. RESULTS: Contact lenses soaked in the MPS induced cell morphology alterations and loss of cell viability. Rinsing the lens with the marine solution improved cell viability and preincubating cells with hyaluronic acid inhibited apoptosis. CONCLUSIONS: MPS can be damaging for the ocular surface cells. We proposed to rinse the lens with a marine solution before insertion of the lens on the cornea to wash away the multipurpose solution and to use hyaluronic acid to protect the ocular surface cells against apoptosis induced by MPS.