Human mmp 9 duoset elisa

The secretion of matrix metalloproteinase 9 (MMP9) into NHEK culture medium was determined using a Human MMP-9 DuoSet ELISA (Cat# DY911, R&D Systems, Minneapolis, MN, USA) and was performed according to the manufacturer’s instructions.

To measure MMP-2 and MMP-9 secretion, enzyme-linked immunosorbent assay (ELISA) was conducted using effluent samples collected from implantation-on-a-chip devices maintained in two different culture conditions: EVT monoculture (EVT-mono) and EVT-endothelial coculture (CO). Three devices were prepared for each condition and cultured for 6 days with media exchange every other day. At Day 6, 400 µl of media was pipetted manually from each chamber through the open medium reserovoirs and combined into an eppendorf tube to generate a 800-µl media sample from each device. Cellular debris was removed by centrifugation at 3000 x g for 10 min at 4 °C, and the supernatant was transferred to a new tube. Three replicates of the spent media for each culture condition were prepared and stored at −80 °C until analysis. For this analysis, we used commerically available ELISA kits (Human MMP-2 DuoSet ELISA: R&D Systems, catalog # DY902, Human MMP-9 DuoSet ELISA: R&D Systems, catalog # DY911) following manufacturer’s protocols. The level of each MMP was calculated using standard curves constructed with recombinant proteins.