Tjp1 elisa kit

The CaCo-2 lysates were used to analyze occludin level by Human Occludin (OCLN) ELISA Kit (MyBiosource, San Diego, CA, USA), claudin-1 level by ELISA Kit (Cusabio Technology LCC, Huston, Houston, TX, USA), and Zona Occludens 1 (ZO-1) level by the human tight junction protein 1 (TJP1) ELISA kit (MyBiosource, San Diego, CA, USA) following the manufacturer’s instructions [31 ]. The absorbance was measured by a Tecan spectrophotometer at 450 nm. The data were obtained by comparing it to the standard curve (from 0 to 1500 pg/mL for occludin and from 0 to 1000 pg/mL for both claudin-1 and ZO-1). The results were presented as a percentage (%) compared to the control (0 line), which comprised five separate experiments conducted in triplicate.

The Human Tight Junction Protein 1 (TJP1) ELISA kit (MyBiosource, San Diego, CA, USA) was used to measure the presence of tight junction protein 1 or Zona occludens 1 (ZO1) in cell lysates in Caco-2, following the manufacturer’s instructions. Briefly, the cells were lysed using trypsin and then collected by centrifugation. Then, cells were washed three times with cold PBS 1× and then resuspended in PBS 1×; subsequently, the cells were subjected to ultrasonication four times and then they were centrifuged at 1500× g for 10 min at 4 °C in order to remove cellular debris. A quantity of 100 μL of each sample was added to a well and incubated at 37 °C for 90 min; then, the materials were removed, and to each well was added 100 μL of Detection Solution A and incubated for 45 min at 37 °C. Wells were washed with Wash Solution and after 100 μL Detection Solution B was added to each well. After incubation of 45 min, the wells were washed again and 90 μL of Substrate Solution was added in each well and then incubated for 20 min at 37 °C in the dark. Finally, 50 μL of Stop Solution was added and then the plates were read by a spectrometer (VICTOR × 4, multilabel plate reader) at 450 nm. The concentration is expressed as ng/mL.