af647 antibody labeling kit  (Thermo Fisher)

Journal: iScience

Article Title: Human monocyte-derived macrophages shift subcellular metalloprotease activity depending on their activation state

doi: 10.1016/j.isci.2024.111171

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Article Snippet: MMP-9, BSA and TIMP-1 were fluorescently labeled with Alexa Fluor (AF)546 (Alexa Fluor 546 Antibody Labeling Kit, Cat # A20183, ThermoFisher Scientific) and/or AF647 (Alexa Fluor 647 Antibody Labeling Kit, Cat # A20186, ThermoFisher Scientific) to track their subcellular presence over time by using fluorescence microscopy.

Techniques: Plasmid Preparation, Recombinant, Knock-Out, Blocking Assay, Staining, Antibody Labeling, Reverse Transcription, Lysis, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Fractionation, Cell Culture, Software, Flow Cytometry, Fluorescence, Spectrophotometry

Journal: The Journal of Biological Chemistry

Article Title: A proteomics-based survey reveals thrombospondin-4 as a ligand regulated by the mannose receptor in the injured lung

doi: 10.1016/j.jbc.2024.107284

Figure Lengend Snippet: Uptake of fluorescence-labeled TSP-4 in alveolar macrophages. A , confocal imaging of lungs from Col1a1.GFP transgenic mice subjected to alveolar instillation of AF647-labeled TSP-4 and injection of Hoechst solution (see ). Mice were euthanized, and lungs were examined 24 h after TSP-4 instillation to reveal TSP-4 internalized in vivo ( red ), GFP-positive cells (fibroblasts; green ) and cell nuclei ( blue ). B and C , MR-dependent uptake of TSP-4 in alveolar macrophages ex vivo . Cells contained in BALF samples from WT (MR +/+) and MR-deficient mice (MR −/−) were harvested and presented with AF647-labeled TSP-4. After endocytosis, alveolar macrophages (CD45+ F4/80+ CD11c high cells) of both genotypes were identified using flow cytometry ( B ) and the mean fluorescence intensity for TSP-4 associated with these cells recorded ( C ). More than 95% of all cells were alveolar macrophages in BALF samples from mice of both genotypes. Histograms ( C , left panel) and bar chart ( C , right panel) reveal a marked reduction in the TSP-4 uptake in MR −/− cells as compared with WT. n = 4. ( p = 0.0014; two-tailed student's t test). AM, alveolar macrophages; BALF, bronchoalveolar lavage fluid; MR, mannose receptor; TSP-4, thrombospondin-4.

Article Snippet: The following proteins and reagents were purchased from commercial sources: acid-extracted collagen type I from rat tail (Corning), D-Mannose-BSA (Dextra), recombinant TSPs 1-5, human periostin/OSF-2 (R&D Systems), D(+)-Galactose and D(+)-Mannose, LPS from Escherichia coli O26:B6 (Sigma-Aldrich), Hoechst 33342, and small scale AF647 antibody labeling kit (Thermo Fisher Scientific).

Techniques: Fluorescence, Labeling, Imaging, Transgenic Assay, Injection, In Vivo, Ex Vivo, Flow Cytometry, Two Tailed Test