Mouse IL-10 Antibody

Detection of Recombinant Mouse IL‑10 by Western Blot. Western blot shows 25 ng of Recombinant Mouse IL-10 (Catalog # 417-ML), Recombinant Human IL-10 (Catalog # 217-IL), Recombinant Rat IL-10 (Catalog # 522-RLB), and Recombinant Human IL-26/AK155 Monomer (Catalog # 1375-IL). PVDF Membrane was probed with 1 µg/mL of Rat Anti-Mouse IL-10 Monoclonal Antibody (Catalog # MAB417) followed by HRP-conjugated Anti-Rat IgG Secondary Antibody (Catalog # HAF005). A specific band was detected for IL-10 at approximately 15 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 3.

Cell Proliferation Induced by IL‑10 and Neutralization by Mouse IL‑10 Antibody. Recombinant Mouse IL-10 (Catalog # 417-ML) stimulates proliferation in the MC/9-2 mouse mast cell line in a dose-dependent manner (orange line). Proliferation elicited by Recombinant Mouse IL-10 (2.5 ng/mL) is neutralized (green line) by increasing concen-trations of Rat Anti-Mouse IL-10 Monoclonal Antibody (Catalog # MAB417). The ND₅₀ is typically 0.005-0.015 µg/mL.

Detection of Mouse IL-10 by Western Blot Imipramine downregulates an M2-like program in TAMs(A) Western blot analysis of ARG1 and IL-10 in single tumors treated or not with IM or anti-VEGF.(B) Flow cytometry analysis of ARG1 and IL-10 expression in GBM tumors treated for 1 week. Ctrl (n = 9 tumors), IM (n = 6), anti-VEGF (n = 6), anti-VEGFR2 (n = 6), Axitinib (n = 6). Macrophages were gated as CD45+CD11b+Ly6C-Ly6G−.(C) Expression of ARG1 and IL-10 in microglia (CD49d−) and MDMs (CD49d+) assessed by FACS in untreated tumors (n = 5).(D) Expression of MHC-II within microglia as assessed by flow cytometry. Ctrl (n = 4 tumors) and IM + anti-VEGF (n = 4).(E) Ex vivo co-cultures of tumoral CD11b cells and activated splenic CFSE-labeled CD8 or CD4 T cells. Each dot represents the average of two or three technical replicates. T cells alone (n = 4), Ctrl co-culture (n = 5), anti-VEGF (n = 3), IM (n = 4), IM + anti-VEGF (n = 4).(F) Analysis of the M2-like program in cytokine-polarized macrophages as assessed by qRT-PCR analysis of Ctrl and IM-treated M2-like BMDMs. Expression is normalized to 18S statistics by Welch’s t test. Each dot represents an individual sample. Data are representative of three independent experiments.(G) Analysis of the M1-like program in BMDMs assessed by FACS. Each dot represents an individual replicate.(H) Expression of Hrh1 mRNA normalized to 18S in ex vivo M1-and M2-polarized BMDMs, either untreated or IM treated for 24 h. Each dot represents an individual sample. Data are representative of three independent experiments.(I) mRNA expression of Hrh1 in FACS-sorted microglia or MDMs from Ctrl and IM-treated tumors.(J) mRNA expression of Arg1, Chil3, and Il10 in M2-polarized macrophages that were transfected with siCtrl or two different siHrh1 constructs. Cells were treated with 40 μm IM for 24 h. Data are representative of two independent experiments.(K) Western blot analysis of MRC1 and ARG1 expression of siRNA-transfected M2 BMDMs. Data are representative of two independent experiments.(L) CD8 and CD4 T cell proliferation during co-culture with tumoral CD11b cells isolated from untreated (n = 2) or TFP-treated tumors (n = 4).(M) mRNA expression of Hrh1, Arg1, and MMP2 in CD11b cells isolated from tumors treated with IM (n = 4), TFP (n = 4), or untreated Ctrl (n = 5).(N) Phagocytosis assay involving sorted microglia and MDMs from untreated or IM-treated tumors assayed with green pHrodo S. aureus bioparticles. Data presented as mean fluorescence intensity (MFI) of pHrodo/live cells. (Para break) Data in all quantitative panels are presented as mean ± SEM ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/36113478), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IL-10 by Immunocytochemistry/Immunofluorescence Lymphocyte infiltrate in tumors from IL-10 deficient mice (A, C and D) or from anti-IL10/IL-10R neutralizing antibody treated mice (B, E and F). A. Single cell suspensions were stained with the indicated antibodies plus anti-CD45 and anti-F4/80. Lymphocytes were analyzed in the CD45+F4/80- population. B. After 6 hours incubation in tissue culture flasks, non adherent cells from total tumor suspension were harvested and stained with the indicated antibodies, tetramer and anti-CD45. C and E. Immunofluorescence of cryo-sections from tumors harvested from IL-10 KO and wild type mice or neutralizing anti-IL10/IL-1R and irrelevant IgG antibodies, as indicated. Sections were stained with anti-CD8 (in FITC, green) and PE (red) conjugated HPV16 E749-57 tetramer - E749-57 Tet, and irrelevant tetramer - Irrelevant Tet. Cells were counterstained with DAPI (blue). Arrowheads indicate double positive cells in yellow, arrows indicate cells positive only for CD8. D and F. Granzyme B expression in tumor cryo-sections from IL-10 KO and wild type mice or mice treated with anti-IL10/IL-10R neutralizing antibodies and irrelevant IgG, as indicated. Sections were stained with anti-CD8 (green), followed by fixation, permeabilization and staining with anti-Granzyme B (red). Tissue was counterstained with DAPI. Solid arrows indicate double positive cells, arrowhead indicate CD8 only positive cell. Insets show details of cells positive only for CD8 (green) or double positive CD8/tetramer (C and E), CD8/Granzyme B (D and F) in yellow or orange due to the sobreposition of emission from FITC and PE (tetramer) or alexa 594 (Granzyme B). Image collected and cropped by CiteAb from the following publication (https://bmcimmunol.biomedcentral.com/articles/10.1186/1471-2172-11-27), licensed under a CC-BY license. Not internally tested by R&D Systems.