Quantikine elisa system

Cells were seeded in 6-well plates (2.5 × 10⁵ cells/well) with the respective culture medium. After the cells had attached to the bottom of the plate, the culture medium was washed with phosphate-buffered saline (PBS), and the medium was changed to RPMI 1640 (Hyclone, Logan, UT) without FBS for 48 h. CM was collected and filtered through a 0.2-µm filter. Experiments with human samples were reviewed and approved by the Joint Institutional Review Board of Taipei Medical University (TMU-JIRB 201312052). Serum from patients who were diagnosed with BPH and prostate cancer was obtained from the TMU bio-bank. All patients provided informed consent for the use of their blood samples and clinical data for research. Concentrations of MMP-3 and -10 in CM and patient serum were determined using the Quantikine® ELISA system (R&D Systems) following manufacturer recommendations. The optical density was determined at 450 nm, and readings at 570 nm were subtracted to correct for optical imperfections in the plate by using a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MO).

For quantification of mouse IL13 in culture supernatants of FAPs, the Quantikine ELISA system (R&D systems) was used. FAPs isolated from WT and Tchp^−/− mice were cultured for 1 week and 1 × 10⁵ FAPs were re‐seeded in 12‐well‐plates with siRNA. Six hours after plating, the medium was changed with the fresh medium and incubated for 2 days. The supernatants were stored at −80 °C until ELISA analysis. The optical density of 96‐well culture plates was measured with a microplate reader Infinite F50 (TECAN).

At 24‐h post‐treatment with IL4 the supernatant was retrieved. CCL26 and IL8 concentrations were measured using Quantikine ELISA system (R&D Systems, Abingdon, UK) as per manufacturer's instructions.