) 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. " width="100%" height="100%">
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
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