monoclonal antibodies against murine cd115/c-fms/m-csfr/csf1r clone afs98  (Thermo Fisher)

Journal: Frontiers in Immunology

Article Title: Patrolling monocytes mediate virus neutralizing IgG effector functions: beyond neutralization capacity

doi: 10.3389/fimmu.2025.1600056

Figure Lengend Snippet: Contribution of patrolling monocytes for antiviral activity of Wen3. (A) Schematic presentation of the experiments in (B-E) . (B) Flow cytometric analysis of monocyte subsets in the peripheral blood and spleen of control or clodronate liposomes-treated mice 3 days post infection (d.p.i.). Animals were treated with 0.5 µL/g control or clodronate liposomes (Clo. (low) ) on day -1 and 1. Mice were infected with LCMV-WE (2x10 5 PFU) on day 0. See ( Supplementary Figure S2 ) for gating strategy of the monocyte subsets. (C) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, and lymphocytes/macrophages in the peripheral blood and spleen of control or clodronate liposome-treated mice 3 d.p.i. Animals were treated with 0.5µL/g control or clodronate liposomes (Clo. (low) ) on day -1 and 1. mice were infected with LCMV-WE (2x10 5 PFU) on day 0. Results show the pooled data from two independent experiments with similar results. (n=3–5 mice/group/experiment). (D) Virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), On day -1 and day 1 mice were treated with 0.5µL/g bodyweight control or clodronate liposomes (Clo. (low) ). Results show the pooled data from three independent experiments with similar results (n=3–4 mice/group/experiment). (E) Spleen sections collected 3 d.p.i. were stained for CD169 (green) and LCMV nucleoprotein (−NP) (red). Shown are the representative merged images from three independent experiments. Scale bar= 300 µm. (F) Virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with neutralizing serum (Neutralizing S.) or naïve serum (see methods). On day -1 and day 1 mice were treated with 0.5µL/g control or clodronate liposomes (Clo. (low) ). Results show the pooled data from two independent experiments with similar results (n=3–5 mice/group/experiment). (G) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, along with lymphocytes in the peripheral blood or macrophages in spleen (3 d.p.i.) of mice treated with anti-CD115 or Isotype. Mice were infected with LCMV-WE (2x10 5 PFU) on day 0, preceded by treatment with 100 µg anti-CD115 or isotype (rat IgG2b) on day -1 and again on day 1. Results show the pooled data from two independent experiments with similar results (n=3 mice/group/experiment). (H) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a). On day -1 and day 1 mice were treated with 100 µg anti-CD115 or isotype. Results show the pooled data from two independent experiments with similar results (n=3 mice/group/experiment). (I) Shown are the virus titres (3 d.p.i.) in spleen of CD11c-cre +/+ /iDTR and CD11c-cre tg/+ /iDTR mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), on day -1 and day 1 mice were treated with diphtheria toxin 12ng/g bodyweight. Results show the pooled data from two independent experiments with similar results (n=3–4 mice/group/experiment). (J) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), mice were treated with 200 µg 9E9 or isotype for blocking FcγRIV. Results show the pooled data from three independent experiments with similar results (n=3–4 mice/group/experiment). (K) Heat map showing the expression of FcγRIV on CD11b + Ly6G - population gated from CD45 + Lin - (CD45R + CD8 + CD4 + ), 24 hour following infection with LCMV. Statistical analysis was performed by using the Student’s t-test (C, G) or One-way ANOVA test (D, F, H–J) . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, ns, non-significant. Horizontal dotted lines indicating the detection limit.

Article Snippet: Depletion antibodies utilized were CD115 (Clone: AFS98), Ly6G (Clone: 1A8), NK (Clone: PK136), all procured from BioXcell, and each was administered at a dosage of 200 μg per mouse (except CD115 100 μg).

Techniques: Activity Assay, Control, Liposomes, Infection, Virus, Staining, Blocking Assay, Expressing

Journal: bioRxiv

Article Title: Sexual dimorphism in melanocyte stem cell behavior reveals combinational therapeutic strategies for cutaneous repigmentation

doi: 10.1101/2023.05.22.541644

Figure Lengend Snippet: ( A ) UMAP of immune cell clusters from WT_Ctrl, WT_UV and RP_UV. WT_Ctrl and WT_UV were the same dataset from . RP_UV sample was from a RP male mouse collected at day 5, the same timepoint as WT_UV. ( B ) Barplot showing the composition of each cell cluster within the three samples. ( C ) UMAP of macrophage subclusters isolated from all three samples. Macrophages were subclustered from cluster Macrophage_1–4 and Proliferative macrophages. ( D ) Visualization of the Inflammation gene set expression in macrophage subclusters. ( E ) Violin plot showing expression of the Inflammation gene set in Inflammatory macrophage, Final_RPUV, Final_WTUV, Final_WTCtrl subclusters. ( F ) Visualization of Complement & Phagocytosis gene set expression in macrophage subclusters. ( G ) Schematic timeline for macrophage depletion using clodronate liposomes (CL), and control PBS liposomes (PL). ( H ) Monocyte depletion in the circulation was validated by flow cytometry. Blood monocytes were identified as CD45+CD115+. Ly6C was used to classify infiltrating monocytes (Ly6C hi ). Truncated skin macrophage infiltration by CL depletion was validated by F4/80 staining on sectioned skin collected at day 5. Final melanocyte migrations were quantified by Dct staining on sectioned skin collected at day 13. Migrations were normalized based on individual level of Cox-2 expression in each mouse. n=8 mice for CL (4 male, 4 female), n=17 for PL (9 male, 8 female). Uneven number due to the poor animal survival under CL treatment. ( I ) Schematic of melanocyte proliferation assay on RP-TT mice under macrophage depletion. Doxycycline, tamoxifen and PL/CL treatments follow the timeline. ( J ) Representative images of tdTomato co-labeling with EdU staining (left), and quantification (right). n=5 mice for CL, n=4 mice for PL. Scale bar: 100um. Statistics: Mann-Whitney t-test ( H ), Welch’s t-test ( I ). Error bar: SEM. Each individual dot indicates the averaged quantification from one mouse. *,**,*** indicates p values are lower than 0.05, 0.01, 0.001, respectively.

Article Snippet: The following antibodies and dyes were used: CD45 (clone 30-F11; BD Biosciences Cat# 564279), CD11b (clone M1/70; Thermo Fisher Scientific Cat# 45–0112-80), CD115 (clone AFS98; Thermo Fisher Scientific Cat# 12–1152-81), Ly6C (clone HK1.4; Thermo Fisher Scientific Cat# 47–5932-80), Ly6G (clone 1A8, BD Biosciences Cat# 565369), F4/80 (clone BM8, Thermo Fisher Scientific Cat# 17–4801-80), Live/Dead Aqua or Violet fixable stains (Life: L34964, NC0180395).

Techniques: Isolation, Expressing, Liposomes, Control, Flow Cytometry, Staining, Proliferation Assay, Labeling, MANN-WHITNEY