Quantification of Occludin in CaCo-2 Cells

The Human Occludin ELISA kit (OCLN kit, MyBiosource, San Diego, CA, USA) analyzed the occludin presence in CaCo-2 cell lysates, according to the manufacturer’s instruction [66 (link)]. Briefly, CaCo-2 cells were lysed with cold Phosphate-Buffered Saline (PBS, Merck Life Science, Rome, Italy) 1×, centrifuged at 1500× g for 10 min at 4 °C, and 100 μL of each sample was transferred to the strip well before the incubation at 37 °C for 90 min. The supernatants were removed, and the strips were incubated with 100 μL of Detection Solution A for 45 min at 37 °C; then, the strips were washed with Wash Solution and incubated with 100 μL of Detection Solution B for an additional 45 min. At the end of this time, 90 μL of Substrate Solution was added followed by an incubation for 20 min at 37 °C in the dark, and then 50 μL of Stop Solution was used to block the enzymatic reaction. The plate was analyzed by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 450 nm. The concentration is expressed as pg/mL compared to a standard curve (range from 0 to 1500 pg/mL) and the results are expressed as percentage (%) versus control (0 line).