Human mmp 2 duo set elisa

Heat-inactivated fetal bovine serum (FBS, HyCylone™) and PBS (10X), pH 7.4 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium high glucose 4.5 g/L (DMEM), penicillin-streptomycin solution, trypsin-EDTA (0.25%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS substrate), 3,3′,5,5′-tetramethylbenzidine (TMB substrate), human glycated albumin, elastase from human leukocytes, N-succinyl-Ala-Ala-Ala-p-nitroanilide, oleanolic acid, hyaluronidase from bovine testes, 4 (dimethylamino)benzaldehyde (DMAB), hyaluronic acid sodium salt from cockscomb, sodium aurothiomalate hydrate (SATMH) and Collagenase Activity Colorimetric Assay Kit (MAK293) were purchased from Merck (Waltham, MA, USA). ELISA Human IL-6 Kit (900-K16) and ELISA Human IL-8 Kit (900-K18) were purchased from PeproTech (London, UK). Human Total MMP-1 DuoSet ELISA (DY901B-05), Human MMP-2 Duo-Set ELISA (DY902), and Human Pro-Collagen I alpha 1 DuoSet ELISA (DY6220-05) were purchased from R&D Systems (Minneapolis, MI, USA).

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.