A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

Electroporation or nucleofection yields transfection efficiencies of ~20-40% in primary RPE.

“However, electroporation or nucleofection has been reported to yield transfection efficiencies of ~20–40% (Toops et al., 2014).”

Primary porcine RPE cultures grown on Transwell filters exhibit correct localization of tight junction markers such as ZO-1 and have a TER of ~400 Ω cm2 as early as 2 weeks in culture.

“Primary porcine RPE cultures, grown on Transwell filters, exhibit correct localization of tight junction markers, such as ZO-1, and have a TER of ~400 Ω cm2, as early as 2 weeks in culture (Toops et al., 2014).”

Polarity and differentiation of RPE cultures are influenced by the source of the cells, plating density, and the substrate on which the cells are grown.

“Polarity and differentiation are not only influenced by the source of the cells (transformed, primary, stem cell-derived), but also by plating density (Adijanto and Philp, 2014; Toops et al., 2014), and the substrate on which the cells are grown (Opas, 1989).”

Primary RPE cultures are usually cultured in medium with 1% serum, so serum starvation is not as big a stressor as it would be for RPE cell lines cultured in high serum-containing medium.

“This is likely because primary RPE cultures are usually cultured in medium with 1% serum (Toops et al., 2014), and thus serum starvation is not as big a stressor as it would be for RPE cell lines that are cultured in high serum-containing medium (e.g., 10% fetal bovine serum [FBS] recommended by ATCC to culture ARPE-19 cells).”