Human MMP-9 Antibody

MMP‑9 in NS0 Mouse Cell Line. MMP‑9 was detected in immersion fixed NS0 mouse myeloma cell line transfected with MMP-9 using 5 µg/mL Human MMP‑9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF911) for 3 hours at room temperature. Cells were stained (red) and counterstained (green). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

MMP‑9 in Human Ovarian Cancer Tissue. MMP-9 was detected in immersion fixed paraffin-embedded sections of human ovarian cancer tissue using Human MMP-9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF911) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

MMP‑9 in MCF‑7 Human Cell Line. MMP-9 was detected in immersion fixed MCF-7 human breast cancer cell line using Human MMP-9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF911) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (yellow; Catalog # NL001) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

MMP‑9 in Human Ovarian Cancer Tissue. MMP-9 was detected in immersion fixed paraffin-embedded sections of human ovarian cancer tissue using Human MMP-9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF911) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human MMP-9 by Proximity Ligation Assay Univariate survival analysis of disease free survival (DFS) in plasma for all 465 patients. A) MMP-9:TIMP-1 measured by ELISA. B) MMP-9:TIMP-1 measured by PLA. Patients are divided into four groups of equal size (Q1-Q4) according to increasing plasma MMP-9:TIMP-1 levels; Q1 being the group with the lowest level. Image collected and cropped by CiteAb from the following open publication (https://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-13-598), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human MMP-9 by Proximity Ligation Assay Univariate survival analysis of disease free survival (DFS) in plasma for all 465 patients. A) MMP-9:TIMP-1 measured by ELISA. B) MMP-9:TIMP-1 measured by PLA. Patients are divided into four groups of equal size (Q1-Q4) according to increasing plasma MMP-9:TIMP-1 levels; Q1 being the group with the lowest level. Image collected and cropped by CiteAb from the following open publication (https://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-13-598), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human MMP-9 by Western Blot Co-culture of THP1-derived macrophages with Panc89 hA8 WT and KO cells. (A) The schematic model depicts the interactions of THP1-derived macrophages (green, M0) and Panc89 cells with or without ADAM8 (red). Created with BioRender.com. (B) ADAM8 mRNA expression in both Panc89 hA8 WT and KO is not affected by M0, whereas LCN2 mRNA expression. Data are presented as mean values ± S.D. *** p < 0.001. (C) is upregulated after co-culture in an ADAM8-dependent manner. Data are presented as mean values ± S.D. *** p < 0.001. (D) The graph illustrates the upregulation of MMP-9 mRNA expression in both Panc89 hA8 WT and KO after co-culture (n = 2). Data are presented as mean values ± S.D. *** p < 0.001. (E) Representative immunoblot shows the detection of ADAM8, MMP-9, and LCN2 with or without co-culture. In addition to the qPCR results, MMP-9 and LCN2 are upregulated after co-culture at the protein level (n = 2). (F) Representative zymography of Panc89 hA8 WT and KO cells with or without co-culture demonstrates less active MMP-9 in Panc89 hA8 KO cells than in Panc89 hA8 WT cells after co-culture. (G) Quantification of active MMP-9 refers to total MMP-9 in zymography of Panc89 hA8 WT and KO cells after co-culture (n = 2). Data are presented as mean values ± S.D. * p < 0.05. Representative images of Panc89 cells before and after co-culture are shown in (H); scale bar, 100 μm. After co-culture, morphological changes are visible in both Panc89 hA8 WT and KO cells. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35216088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human MMP-9 by Western Blot ADAM8 and LCN2 levels correlate in Panc89- and MB-231-derived extracellular vesicles (EV). Representative Western blots (A) of EVs derived from either Panc89 hA8 WT or KO cells, and cell lysate (CL) of Panc89 hA8 WT cells, and (B) of EVs derived from either MB-231 hA8 WT or MB-231 KO cells show the detection of ADAM8, MMP-9, LCN2, and Flotillin-1 in the upper part. Diagrams below illustrate the quantification and downregulation of LCN2 secretion (relative to Flotillin-1 secretion) in EVs isolated from Panc89 (hA8 WT or hA8 KO 1) or MB-231 (hA8 WT or hA8 KO) cells (n = 3). Data are presented as mean values ± S.D. ** p < 0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35216088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human MMP-9 by Western Blot Co-culture of THP1-derived macrophages with Panc89 hA8 WT and KO cells. (A) The schematic model depicts the interactions of THP1-derived macrophages (green, M0) and Panc89 cells with or without ADAM8 (red). Created with BioRender.com. (B) ADAM8 mRNA expression in both Panc89 hA8 WT and KO is not affected by M0, whereas LCN2 mRNA expression. Data are presented as mean values ± S.D. *** p < 0.001. (C) is upregulated after co-culture in an ADAM8-dependent manner. Data are presented as mean values ± S.D. *** p < 0.001. (D) The graph illustrates the upregulation of MMP-9 mRNA expression in both Panc89 hA8 WT and KO after co-culture (n = 2). Data are presented as mean values ± S.D. *** p < 0.001. (E) Representative immunoblot shows the detection of ADAM8, MMP-9, and LCN2 with or without co-culture. In addition to the qPCR results, MMP-9 and LCN2 are upregulated after co-culture at the protein level (n = 2). (F) Representative zymography of Panc89 hA8 WT and KO cells with or without co-culture demonstrates less active MMP-9 in Panc89 hA8 KO cells than in Panc89 hA8 WT cells after co-culture. (G) Quantification of active MMP-9 refers to total MMP-9 in zymography of Panc89 hA8 WT and KO cells after co-culture (n = 2). Data are presented as mean values ± S.D. * p < 0.05. Representative images of Panc89 cells before and after co-culture are shown in (H); scale bar, 100 μm. After co-culture, morphological changes are visible in both Panc89 hA8 WT and KO cells. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35216088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human MMP-9 by Western Blot ADAM8 and LCN2 levels correlate in Panc89- and MB-231-derived extracellular vesicles (EV). Representative Western blots (A) of EVs derived from either Panc89 hA8 WT or KO cells, and cell lysate (CL) of Panc89 hA8 WT cells, and (B) of EVs derived from either MB-231 hA8 WT or MB-231 KO cells show the detection of ADAM8, MMP-9, LCN2, and Flotillin-1 in the upper part. Diagrams below illustrate the quantification and downregulation of LCN2 secretion (relative to Flotillin-1 secretion) in EVs isolated from Panc89 (hA8 WT or hA8 KO 1) or MB-231 (hA8 WT or hA8 KO) cells (n = 3). Data are presented as mean values ± S.D. ** p < 0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35216088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human MMP-9 by Western Blot Co-culture of THP1-derived and polarized macrophages with Panc89 hA8 WT and KO cells. (A) Western blot illustrates the detection of ADAM8, MMP-9, and LCN2 in Panc89 hA8 WT and KO control cells (Ø) and after co-culture with M0, M1, and M2 macrophages (two time points: 0 h and 1 h). ADAM8 is upregulated in Panc89 hA8 WT cells after co-culture with M2-polarized macrophages. Panc89 cells show the highest MMP-9 expression after co-culture with M0, but M1 macrophages also upregulate MMP-9. LCN2 is dependent on ADAM8 when upregulated in Panc89 cells after co-culture with M0 and M2 macrophages but independent of ADAM8 in Panc89 cells co-cultured with M1 macrophages. (B) ADAM8, (C) MMP-9, and (D) LCN2 ELISA of Panc89 hA8 WT and KO cell-derived supernatants of control cells and after co-culture with M0, M1, and M2 (two time points: 0 h and 1 h). In accordance with the immunoblot results of (A), ADAM8 is upregulated in supernatants derived from Panc89 hA8 WT cells after co-culture with M2 macrophages (B). At the same time, macrophages increase MMP-9 secretion from an undetectable level to almost 80,000 pg/mL in Panc89 hA8 WT and 60,000 pg/mL in Panc89 hA8 KO cells. M1 and M2 macrophages increase MMP-9 secretion of Panc89 independent of ADAM8, but not as high as in Panc89 cells co-cultured with M0. In contrast, LCN2 is upregulated in Panc89 hA8 WT cells by M0 and M1, but not by M2 macrophages. In the absence of ADAM8, Panc89 hA8 KO cells show low LCN2 secretion in control cells and after co-culture with M0 and M2 macrophages. Only after co-culture with M1 macrophages is the LCN2 secretion level increased (n = 1). Data are presented as mean values ± S.D. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35216088), licensed under a CC-BY license. Not internally tested by R&D Systems.