Human Myeloperoxidase/MPO Antibody

Detection of Human Myeloperoxidase/MPO by Western Blot. Western blot shows lysates of HL-60 human acute promyelocytic leukemia cell line and human neutrophil. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human Myeloperoxidase/MPO Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3174) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). A specific band was detected for Myeloperoxidase/MPO at approximately 60-65 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

Myeloperoxidase/MPO in HL‑60 Human Cell Line. Myeloperoxidase/MPO was detected in immersion fixed HL‑60 human acute promyelocytic leukemia cell line (positive staining) and HDLM‑2 human Hodgkin’s lymphoma cell line (negative staining) using Goat Anti-Human Myeloperoxidase/MPO Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3174) at 1.7 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. Staining was performed using our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Human Myeloperoxidase/MPO by Simple Western^TM. Simple Western lane view shows lysates of human neutrophils, loaded at 0.2 mg/mL. A specific band was detected for Myeloperoxidase/MPO at approximately 65 kDa (as indicated) using 5 µg/mL of Goat Anti-Human Myeloperoxidase/MPO Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3174) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Mouse Myeloperoxidase/MPO by Immunocytochemistry/Immunofluorescence Cathelicidins induce platelet–neutrophil interactions. a–h Co-incubation experiments. Human platelets were pretreated with LL-37 or scrambled control peptide (Scra) and platelet–neutrophil interactions were analyzed. a–e Flow cytometry analysis of a platelet–neutrophil aggregates formation (n = 9), b platelet–neutrophil aggregates in the presence of a blocking antibody against P-selectin and respective isotype control (n = 5), c CD11b expression on neutrophils, d neutrophil intracellular formation of reactive oxygen species (ROS), e shedding of neutrophil L-selectin (n = 4). TNF alpha (50 ng/mL) served as positive control. f–h Neutrophil extracellular trap (NET) formation assay. f Representative epifluorescence image of a NET. DAPI (nuclear stain, blue), myeloperoxidase (MPO, red), and citrullinated histone H3 (citH3, green). Bar, 10 µm. g NET formation was induced by platelets that were pretreated with LL-37 or a GPVI-activating antibody (HGP4C9). Upper row (DAPI nuclear stain, white), middle row (MPO, red), and bottom row (merged image of DAPI in blue, and MPO in red). Arrowheads indicate NET. Bar, 10 µm. h Quantitative analysis of NET formation (n = 4). i, j Interactions of mouse cells. i Platelet–neutrophil aggregates formation of mouse neutrophils with platelets isolated from wild type (WT) or P-selectin deficient mice (n = 7). j Platelet–neutrophil aggregates formation after co-incubation of isolated WT platelets with PMA (50 µmol/L) activated neutrophils of WT or CRAMP−/− mice (n = 4). Graphs show mean and SEM. P-values were determined by one-way repeated measures ANOVA with Bonferroni correction (a–c), paired t-test (d, e), ANOVA on Ranks/Dunn’s method (h) or Mann–Whitney U-test (i, j) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29670076), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Myeloperoxidase/MPO by Immunocytochemistry/Immunofluorescence Cathelicidins induce platelet–neutrophil interactions. a–h Co-incubation experiments. Human platelets were pretreated with LL-37 or scrambled control peptide (Scra) and platelet–neutrophil interactions were analyzed. a–e Flow cytometry analysis of a platelet–neutrophil aggregates formation (n = 9), b platelet–neutrophil aggregates in the presence of a blocking antibody against P-selectin and respective isotype control (n = 5), c CD11b expression on neutrophils, d neutrophil intracellular formation of reactive oxygen species (ROS), e shedding of neutrophil L-selectin (n = 4). TNF alpha (50 ng/mL) served as positive control. f–h Neutrophil extracellular trap (NET) formation assay. f Representative epifluorescence image of a NET. DAPI (nuclear stain, blue), myeloperoxidase (MPO, red), and citrullinated histone H3 (citH3, green). Bar, 10 µm. g NET formation was induced by platelets that were pretreated with LL-37 or a GPVI-activating antibody (HGP4C9). Upper row (DAPI nuclear stain, white), middle row (MPO, red), and bottom row (merged image of DAPI in blue, and MPO in red). Arrowheads indicate NET. Bar, 10 µm. h Quantitative analysis of NET formation (n = 4). i, j Interactions of mouse cells. i Platelet–neutrophil aggregates formation of mouse neutrophils with platelets isolated from wild type (WT) or P-selectin deficient mice (n = 7). j Platelet–neutrophil aggregates formation after co-incubation of isolated WT platelets with PMA (50 µmol/L) activated neutrophils of WT or CRAMP−/− mice (n = 4). Graphs show mean and SEM. P-values were determined by one-way repeated measures ANOVA with Bonferroni correction (a–c), paired t-test (d, e), ANOVA on Ranks/Dunn’s method (h) or Mann–Whitney U-test (i, j) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29670076), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Myeloperoxidase/MPO by Immunocytochemistry/Immunofluorescence Cathelicidins are present in human and mouse arterial thrombi. a, b Representative images of coronary artery thrombi isolated from five patients with acute myocardial infarction. a Immunohistochemistry for LL-37 indicated enrichment within leukocytes (arrowhead), but also stained leukocyte-free areas (asterisk). Bars, 200 µm (overview) and 10 µm (magnification). b Immunofluorescence analysis of LL-37 (red), myeloperoxidase (MPO, yellow), CD41 (platelets, green), and DAPI (nuclei, blue). Bar, 10 µm. c, d Representative images of murine carotid artery thrombi generated by ferric chloride injury. c Immunohistochemistry for cathelicidin-related antimicrobial peptide (CRAMP, mouse homologue for LL-37) indicated enrichment within leukocytes (arrowhead), but also stained leukocyte-free areas (asterisk). Bars, 10 µm. d Immunofluorescence analysis of CRAMP (red), CD41 (platelets, green), and DAPI (nuclei, blue). Bar, 10 µm. e Analysis of CRAMP binding in arterial thrombosis in vivo. 5-FAM-labeled CRAMP or scrambled control was injected into wild type mice before induction of ferric chloride injury. Platelets were labeled in vivo using a DyLight649-labeled non-blocking GPIb beta antibody. Left: 5-FAM-labeled CRAMP (green) associated with platelets (GPIb, red in merged image) in the forming thrombus. Right: Image for 5-FAM-labeled control peptide and platelets (GPIb, red in merged image). Bar, 500 µm. See also Supplemental Movies 1, 2. f Flow cytometry analysis of LL-37 binding to isolated human, platelets in vitro. 5-FAM-labeled LL-37 (red), scrambled 5-FAM-labeled control peptide (blue), or vehicle (gray). Graph shows mean and SEM. P-value was determined by unpaired t-test Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29670076), licensed under a CC-BY license. Not internally tested by R&D Systems.