Assessing Tight Junctions in ARPE-19 Cells

Immunofluorescence against zonula occludens-1 (ZO-1) was performed to assess the effect of both N and ND treatments on intercellular tight junctions. ARPE-19 cells were seeded at a density of 100,000 cells per well on laminin-coated polycarbonate membrane cell culture inserts (Corning Life Science, Tewksbury, MA, USA) and were grown in 1% serum-free DMEM for 4 weeks. Immunofluorescence was then performed using a ZO-1 anti-rabbit Alexa Fluor 594 antibody (1:100, 339194, Invitrogen- Life Technologies, Carlsbad, CA, USA) diluted in blocking buffer following the same protocol as previously described [36 (link)]. DAPI (6-diamidino-2-phenylindole; Sigma-Aldrich) was used to stain cell nuclei. Images were obtained with a laser scanning confocal imaging system (LSM800, Zeiss, Oberkochen, Germany). N and ND treatments (62.34 µg/mL) were added individually to the cell line in order to be compared to the saline group, and were also added with either H₂O₂ (1600 µM) or LPS (as described in Section 2.3) in order to identify potential recovery effects on cell integrity.