Human mmp1 elisa kit

Stable MDCK-mmp1 colonies were analyzed for MMP1 secretion levels using a human MMP1 ELISA kit (RayBio). Media collected from various MDCK-mmp1 colonies were assayed directly in the plate following manufacturer’s protocol. Briefly, 100 μL of standard or sample were added to each well and incubated at room temperature for 2 ½ h. Wells were washed and hybridized with the provided antibody solution. After 1 h incubation, wells were washed again and then developed using HRP-streptavidin and TMB substrate solution. OD values were recorded at 450 nm. Three MDCK-mmp1 colonies of varying secretion levels and a mock-transfected MDCK colony were selected for analysis of cellular adhesion to tissue-culture treated plates. Three hours before the shear chamber assay, cells were seeded at 4 x 10⁵ cells per dish in 35mm x 10 mm cell culture dishes. A total of 15 dishes were prepared for each subclone. After visually verifying the adhesion of cells to the surface, cells were washed with Hanks’-based cell dissociation buffer (Gibco) for 5 min at 37°C. After washing, cells were subjected to shear stress at 16 and 24 dyn/cm² using the washing buffer as the moving fluid. The numbers of cells within the image frame were counted before and after exertion of stress using the ImageJ software. For each sub clone and each shear stress level, triplicates of samples were assayed.

Protein levels of COX2 were analyzed in macrophage cell lysates using a commercially available detection kit (DYC4198-2; human/mouse total COX2 ELISA with DuoSet, R&D Systems Europe, Abingdon, United Kingdom). Protein levels of visfatin (human NAMPT ELISA Kit, SK00121-01, Hölzel Diagnostika, Cologne, Germany), TNFα (human TNF-α Quantikine ELISA Kit, DTA00C, R&D Systems, Wiesbaden-Nordenstadt, Germany), and MMP1 (human MMP-1 ELISA Kit, RayBiotech, Norcross, GA, USA) were measured in macrophage cell culture supernatants. The protocols were followed according to the manufacturer’s instructions for each specific kit. The concentration of visfatin, COX2, TNFα, and MMP-1 was measured by spectrophotometry using a microplate reader (PowerWave X, BioTek Instruments, Winooski, VT, USA). Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific) was used to measure the total protein concentrations and normalize the data.