Journal: EMBO Reports

Article Title: Monocytes differentiate along two alternative pathways during sterile inflammation

doi: 10.15252/embr.202256308

Figure Lengend Snippet: CD45.2 + monocytes were injected in CD45.1 + recipient mice previously injected with thioglycolate. Peritoneal lavage was analyzed at 3, 9, 24, 48, and 72 h post transfer ( n = 4 biological replicates). UMAP displaying CD45.2 + Lineage − Ly6G − Tim4 − peritoneal cells analyzed by flow cytometry and concatenated from all time points. UMAP of cells from each time point. Expression of phenotypic markers used for UMAP projection. Supervised gating strategy for moDC and moMac at each time point (gated on live CD11b + CD115 + cells). Supervised gating strategy to identify monocyte‐derived cells. Distribution of manual gates in the UMAP projection. Unsupervised clustering using Phenograph. Heatmap of median marker expression values for each Phenograph cluster. Trajectory and pseudotime analysis using Slingshot. Cluster 12 (monocytes) was chosen as starting point. Trajectories from monocytes towards moDC or moMac are highlighted. Cells are colored by pseudotime.

Article Snippet: Antibodies used were anti‐CD115 BUV 395 (BD Bioscience, clone AFS98), anti‐CD172a BUV737 (BD Biosciences, clone P84), anti‐Ly6G BV510 (Biolegend, clone 1A8), anti‐CD19 BV480 (BD Bioscience clone 1D3), anti‐TCRβ BV480 (BD Bioscience, clone H57‐597), anti‐NK1.1 BV480 (BD Bioscience, clone PK136), anti‐SiglecF BV480 (BD Bioscience, clone E50‐2440), anti‐Ly6G BV480 (Biolegend, clone 1A8), anti‐Ly6G BV605 (Biolegend, clone 1A8), anti‐MHC II BV650 (Biolegend, clone M5/114.15.2), anti‐CCR2 BV711 (BD Bioscience, clone 475,301), anti‐CD11c BV786 (Biolegend, clone N418), anti‐Ly6C BV786 (Biolegend, clone HK1.4), anti‐Ly6C FITC (Biolegend, clone HK1.4), anti‐CD45.2 APC (Biolegend, clone 104), anti‐CD26 PE (Biolegend, clone H194‐112), anti‐CD226 PE (Biolegend, clone 10E5), anti‐CD11b PE da594 (BD Bioscience, clone M1/70), anti‐CD117 PE dazzle594 (Biolegend, clone 2B8), anti‐CD11b PE (BD Biosciences, clone M1/70), anti‐CD11b PerCPCy5.5 (BD Biosciences, clone M1/70), anti‐F4/80 PECy7 (Biolegend, clone BM8), anti‐CD16/32 PECy7 (Biolegend, clone 93), anti‐CD64 PeCy7 (BD Bioscience, clone 10.1), anti‐CD11c PeCy7 (BD Bioscience, clone N418), anti‐MerTK PECy7 (Biolegend, clone 2B10C42), anti‐ESAM APC (Biolegend, clone 1G8/ESAM), anti‐CD115 APC (BD Bioscience, clone AFS98), anti‐CD115 APC‐Fire750 (BD Bioscience, clone AFS98), anti‐TIM4 BB700 (Biolegend, clone RMT4‐54), anti‐CD43 BB700 (BD Bioscience, clone S7), anti‐CD135 APC (Biolegend, clone A2F10), anti‐Ly6C Alexa 700 (Biolegend, clone HK1.4), anti‐MHC II APC Cy7 (Biolegend, clone M5/114.15.2) and anti‐ICAM2 FITC (Biolegend, clone 3C4).

Techniques: Injection, Flow Cytometry, Expressing, Derivative Assay, Marker

Journal: Journal for Immunotherapy of Cancer

Article Title: p38 MAPK signaling in M1 macrophages results in selective elimination of M2 macrophages by MEK inhibition

doi: 10.1136/jitc-2020-002319

Figure Lengend Snippet: M2 macrophages are highly sensitive to MEK inhibition in vivo and ex vivo. (A) Immunohistochemistry of a PDA30364 tumor using an F4/80-specific antibody. Scale bar: 100 µm. (B) Flow cytometric identification of iNOS+ M1 and CD206+ M2 macrophages in a freshly dissociated tumor sample upon gating on the live CD45+, CD11b+, F4/80+ cell subset. (C) MHC-II (IA/IE) and CD86 surface levels of macrophages gated in (B). Mean±SEM, n=4. (D) Mice bearing PDA30364 tumors were treated with MEKi 30 mg kg −1 GDC-0623 (daily), anti-CSF1R antibody (1 mg once, followed by 0.5 mg every other day), or anti-CD40 antibody (200 µg on days 10, 12, 14, and 17 after tumor inoculation). Mean±SEM, n=5. Two-way ANOVA with post hoc Tukey test. (E) Flow cytometry-based quantification of M2 (CD206 + ) and M1 (iNOS + ) macrophages in PDA30364 tumors from mice treated as described in (C). Mean±SEM, n=4. One-way ANOVA with post hoc Dunnett test. (F) Dose–response curves of PDA30364 and M1 (20 ng mL −1 M-CSF+1 ng mL −1 IFNγ/LPS) and M2 (20 ng mL −1 M-CSF+2.5 ng mL −1 IL-4) polarized murine macrophages treated with GDC-0623. Mean±SEM, n=3. Numbers indicate best-fit inhibitory concentration (IC)50 values. (G) Viability of PDA30364 and murine M1 and M2 macrophages treated with GDC-0623 at 1 µM. Mean±SEM, n>4. One-way ANOVA with post hoc Tukey test. Significance levels are indicated by asterisks (*p≤0.05; **p≤0.01; ***p≤0.001: ****p≤0.0001). ANOVA, analysis of variance; IFNγ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; M-CSF, macrophage-colony stimulating factor; MEK, mitogen-activated protein kinase kinase.

Article Snippet: MEKi GDC-0623 (A-1181; Chemgood), p38 MAPKi BIRB 796 (S1574; DP, Selleckchem), murine multimeric CD40L (AG-40B-0020; Adipogen), poly I:C (tlrl-pic; InvivoGen), lipopolysaccharide (LPS) (L4391; Sigma), recombinant murine interferon-γ (IFNγ) (12343536; ImmunoTools), murine macrophage-colony stimulating factor (M-CSF) (12343115; ImmunoTools), murine interleukin (IL)-4 (12340042; ImmunoTools), murine IL-10 (12340105; ImmunoTools), anti-mouse CSF1R (BP0213; BioXCell), recombinant human M-CSF (11343115; ImmunoTools), human IFNγ (11343536; ImmunoTools), human IL-4 (11340045; ImmunoTools), human transforming growth factor-β1 (TGFβ1) (11343160; ImmunoTools), and human IL-10 (11340103; ImmunoTools).

Techniques: Inhibition, In Vivo, Ex Vivo, Immunohistochemistry, Flow Cytometry, Concentration Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Immune signatures of megakaryocytes in persistent inflammation-immunosuppression and catabolism syndrome

doi: 10.3724/abbs.2025087

Figure Lengend Snippet: MKs in the peripheral blood, bone marrow, and spleen of PICS and sepsis model mice and control sham mice (A) Schematic diagram showing the timeline of the experimental design. (B,C) Proportions of monocytes, B cells, plasma cells, and MKs in (B) PICS and (C) sepsis model mice after injection with anti-CD115, anti-B220, or anti-CD25 antibodies (n = 5–6 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant by one-way ANOVA. The error bars represent the range of the changes. (D) The proportions of MKs in the bone marrow and spleen of PICS model mice and sham mice. (E) CD41 staining of MKs in the bone marrow of PICS model mice and sham mice. (F) Size of two representative spleens from sham and PICS model mice. The error bars represent the SDs.

Article Snippet: To deplete specific cell types, PICS mice were injected intraperitoneally (i.p.) with anti-mouse CSF1R (CD115) (clone AFS98, which is usually used as a marker of bone marrow cells, such as monocytes, macrophages, and DCs) [ – ], anti-mouse B220 (clone AFS98, anti-B cell) , or anti-mouse CD25 (clone AFS98, anti-Treg) monoclonal antibodies (BioXCell, West Lebanon, USA).

Techniques: Control, Clinical Proteomics, Injection, Staining

Journal: bioRxiv

Article Title: Macrophage Antigen Presentation Is Unleashed by Pan-RAS Inhibition to Promote Antitumor Immunity

doi: 10.64898/2026.01.30.702886

Figure Lengend Snippet: A) Experimental scheme for B16-OVA transplantation in nude mice followed by RMC-6236 oral gavage (10 mg/kg, daily) ; B) C omparison of tumor size between vehicle and RMC-6236–treated nude mice ; C) Tumor growth curves ; D,E) Flow-cytometry analysis of TAM CD206 (D) and CD86 (E) with vehicle or RMC-6236 ; F) Immunofluorescence showing macrophages (F4/80, green) and MHC-I (red) with DAPI (blue) ; G–I) Flow-cytometry quantification in TAMs of MHC-I (H-2K b ) (G) , OVA-specific presentation (SIINFEKL) (H) , and MHC-II (I-A/I-E) (I); J) Schematic of anti-CSF1R– mediated monocyte/macrophage depletion during RMC-6236 treatment; K) Tumor growth with RMC-6236 plus IgG or anti-CSF1R ; L, M) Intratumoral CD8 + T-cell frequency after monocyte depletion ; N,O) Granzyme B (N) and perforin (O) in tumor CD8 + T-cells. Data are shown as mean ± SEM. *p < 0.05, **p< 0.01, ***p < 0.001 and ****p < 0.0001 using unpaired two-tailed t-tests and Two-way ANOVA followed by Sidak’s multiple comparisons test (n≥6).

Article Snippet: To deplete monocytes/macrophages, mice were treated with 300 μg anti-CSF1R antibody (clone AFS98, Bio X Cell, Cat. No. BP0213) via intraperitoneal injection every 3 days for the indicated pre-treatment period before tumor challenge , .

Techniques: Transplantation Assay, Flow Cytometry, Immunofluorescence, Two Tailed Test

Journal: Brain, Behavior, and Immunity

Article Title: Dorsal root ganglia CX3CR1 expressing monocytes/macrophages contribute to arthritis pain

doi: 10.1016/j.bbi.2022.09.008

Figure Lengend Snippet: Classical and non-classical monocytes and neutrophils infiltrate WT and KO hind paws at day 5 following K/BxN serum transfer. (A) Scatter plots of gating strategy for monocytes and neutrophils. Viable cells were gated using L/D dye and SSC-A: non-classical monocytes were identified as CD45 + CD11b + CD115 + Ly6C low CD43 + CCR2 - , classical monocytes as CD45 + CD11b + CD115 + Ly6C high CD43 - CCR2 + and neutrophils as CD45 + CD11b + CD115 - Ly6G + . (B, C, D) Bar charts present number of classical monocytes, non-classical monocytes and neutrophils in WT and KO hind paws. Data are mean ± S.D.; n = 4 animals per group. *p < 0.05, **p < 0.01, ***p < 0.05, same group, day comparisons # p < 0.05, ## p < 0.01, ### p < 0.001 same day, group comparisons, two-way ANOVA with Tukey multiple-comparison test.

Article Snippet: Cells were first incubated with anti-CD16/CD32 (eBioscience, 1:1000, 15 min, 4) antibody to block unspecific Fc receptor binding and then stained with Fixable Viability Dye eFluorTM 780 (Thermofisher, 1:1000) to identify live cells, while distinct leukocyte subtypes were identified using the following antibodies: CD45 BV450 (0.5 μg/ml, clone 30-F11, BD Biosciences), CD11b PE-Cy7(0.2 ug/mL, clone M1/70, ThermoFisher scientific), CD115 APC (2 μg/ml, clone AFS98, Biolegend), Ly6C (1 μg/ml, clone HK1.4, ThermoFisher scientific), Ly6G PE (0.5 μg/ml, clone 1A8, BD Bioscience), CD43 BV510 (1 mg/ml, clone S7, BD Bioscience), MHCII AF700 (1.25 μg/ml, clone M5/114, Biolegend), F4/80 BV650 (2 μg/ml,clone BM8, ThermoFisher scientific), CCR 2 BV711 (0.5 μg/ml, clone 475301, BD Bioscience), CD206 BV605 (3 μg/ml, clone C068C2, Biolegend).

Techniques: Comparison

Journal: Brain, Behavior, and Immunity

Article Title: Dorsal root ganglia CX3CR1 expressing monocytes/macrophages contribute to arthritis pain

doi: 10.1016/j.bbi.2022.09.008

Figure Lengend Snippet: Olcegepant treatment is associated with less leukocyte infiltration in the paw in K/BxN serum transfer inflammatory arthritis. Within the paw cells suspension, classical monocytes were defined as CD45 + CD11b + CD115 + Ly6C High CD43 - CCR2 + cells, non-classical as CD45 + CD11b + CD115 + F4/80 - Ly6C Low CD43 + CCR 2 - , while neutrophils were CD45 + CD11b + CD115 - Ly6G + . Macrophages were defined as CD45 + CD11b + F4/80 + Ly6C - cells, with CD206 and MHCII staining used to define M1-like (CD206 - MHCII + ) and M2-like (CD206 + MHCII - ) phenotypes. (A-C) Bar charts present classical, non-classical monocytes and neutrophils in control serum, K/BxN serum, and K/BxN serum plus olcegepant paws. (D-F) Bar charts present total macrophages (F4/80 + ), M1-like macrophages (CD206 - MHCII + ), and M2-like macrophages (CD206 + MHCII - ) in the same groups. Data are mean ± S.D.; n = 7–8 animals per group. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Kruskal-Wallis test.

Article Snippet: Cells were first incubated with anti-CD16/CD32 (eBioscience, 1:1000, 15 min, 4) antibody to block unspecific Fc receptor binding and then stained with Fixable Viability Dye eFluorTM 780 (Thermofisher, 1:1000) to identify live cells, while distinct leukocyte subtypes were identified using the following antibodies: CD45 BV450 (0.5 μg/ml, clone 30-F11, BD Biosciences), CD11b PE-Cy7(0.2 ug/mL, clone M1/70, ThermoFisher scientific), CD115 APC (2 μg/ml, clone AFS98, Biolegend), Ly6C (1 μg/ml, clone HK1.4, ThermoFisher scientific), Ly6G PE (0.5 μg/ml, clone 1A8, BD Bioscience), CD43 BV510 (1 mg/ml, clone S7, BD Bioscience), MHCII AF700 (1.25 μg/ml, clone M5/114, Biolegend), F4/80 BV650 (2 μg/ml,clone BM8, ThermoFisher scientific), CCR 2 BV711 (0.5 μg/ml, clone 475301, BD Bioscience), CD206 BV605 (3 μg/ml, clone C068C2, Biolegend).

Techniques: Suspension, Staining, Control