Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ChemR23, the receptor for chemerin and resolvin E1, is expressed and functional on M1 but not on M2 macrophages.

doi: 10.4049/jimmunol.1402166

Figure Lengend Snippet: FIGURE 5. Repolarization of LPS-stimulated primary human M1 macrophages with RvE1 leads to a distinct macrophage phenotype with properties of proresolution macrophages. LPS-stimulated primary human macrophages were repolarized for 4 d in the presence of medium (med), IL-4, or 10 nM RvE1, and mRNA and protein expression of cytokines and cell surface markers were measured to characterize polarization of macrophages. (A–D) mRNA levels of (A) ChemR23, (B) IL-1b, (C) TNF-a, and (D) IL-10 are presented. All mRNA values were normalized for GAPDH and are presented relative to macrophages repolarized with medium alone (med). (E–G) Membrane expression of (E) ChemR23, (F) CD80, and (G) CD206 is shown, whereas in (H), protein secretion of IL-10 into the supernatant is shown. Bars indicate the mean of three independent experiments. *p , 0.05, **p , 0.01.

Article Snippet: Chemerin Receptor 23, anti-human, mAb (ChemR23 Ab) and isotype IgG3, PE-labeled anti-human CD80, monoclonal allophycocyanin-labeled anti-human CD206 Ab, and the IgG1K isotype control were purchased from R&D Systems Europe.

Techniques: Expressing, Membrane

Journal: The Tohoku journal of experimental medicine

Article Title: C-reactive protein reduces the relative number of tumor-associated M2 macrophages and intratumoral angiogenesis in mice.

doi: 10.1620/tjem.233.249

Figure Lengend Snippet: Fig. 2. Macrophage identification. Photomicrographs of F4/80+ cells, which represent the total macrophage population in the CRP group (A) and the con- trol group (B). CD206+ cells represent the M2 macrophage population in the CRP group (C) and the control group (D).

Article Snippet: M2 macrophages were identified using a rat monoclonal anti-CD206 antibody (1:200 dilution, MCA2235; AbD Serotec).

Techniques: Control

Journal: The Tohoku journal of experimental medicine

Article Title: C-reactive protein reduces the relative number of tumor-associated M2 macrophages and intratumoral angiogenesis in mice.

doi: 10.1620/tjem.233.249

Figure Lengend Snippet: Fig. 3. Changes in the size of the macrophage fractions. Group data showing that tumors from CRP-treated mice contain significantly larger numbers of F4/80+ macrophages (A), whereas there is no significant difference in the CD206+ macrophage counts between CRP-treated and control mice (B). Consequently, tumors from CRP-treated mice showed significantly smaller M2 macrophage fractions (C). N.S., not sig- nificant.

Article Snippet: M2 macrophages were identified using a rat monoclonal anti-CD206 antibody (1:200 dilution, MCA2235; AbD Serotec).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: EZH2 Inhibitors Suppress Colorectal Cancer by Regulating Macrophage Polarization in the Tumor Microenvironment

doi: 10.3389/fimmu.2022.857808

Figure Lengend Snippet: | EPZ6438 decreased the infiltration proportion of M2 macrophages but GSK126 increased it in vivo . (A) The statistical graph of flow cytometry on CD11b + F4/80 + cell ratio in CD45 + cells representing total macrophages in the tumor microenvironment. (B) The ratio of CD86 + cells in CD11b + F4/80 + cells representing M1 macrophages. (C) The ratio of CD206 + cells in CD11b + F4/80 + cells representing M2 macrophages. (D) Opal multiplex immunofluorescence staining identified the proportion and location of M1 and M2 macrophages in the tumor microenvironment. Individual staining images of DAPI, F4/80, CD206, CD86, separately (left) and the merged images (right). (E–G) The quantitative analysis regarding the Opal multiplex immunofluorescence staining as shown above. (H) The level of H3K27me3 on the promoter of STAT3 was analyzed by the ChIP-qPCR experiment. The level of H3K27me3 was decreased after GSK126 and EPZ6438 treatment. The experimental results were normalized to a 2% input sample group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance.

Article Snippet: Five-micrometer formalin-fixed, paraffin-embedded (FFPE) tumor sections of the mouse model were stained using a reference protocol ( ) and the panel contained antibodies against DAPI (Cat # : 44010A, BestBio), CD206 (1:500, dye 620, Cat # : 24595, Cell Signaling Technology), CD86 (1:500, dye 690, Cat # : 19589, Cell Signaling Technology), and F4/80 (1:500, dye 540, Cat # :70076, Cell Signaling Technology).

Techniques: In Vivo, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining, ChIP-qPCR

Journal: Frontiers in Immunology

Article Title: EZH2 Inhibitors Suppress Colorectal Cancer by Regulating Macrophage Polarization in the Tumor Microenvironment

doi: 10.3389/fimmu.2022.857808

Figure Lengend Snippet: Primer sequences.

Article Snippet: Five-micrometer formalin-fixed, paraffin-embedded (FFPE) tumor sections of the mouse model were stained using a reference protocol ( ) and the panel contained antibodies against DAPI (Cat # : 44010A, BestBio), CD206 (1:500, dye 620, Cat # : 24595, Cell Signaling Technology), CD86 (1:500, dye 690, Cat # : 19589, Cell Signaling Technology), and F4/80 (1:500, dye 540, Cat # :70076, Cell Signaling Technology).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: EZH2 Inhibitors Suppress Colorectal Cancer by Regulating Macrophage Polarization in the Tumor Microenvironment

doi: 10.3389/fimmu.2022.857808

Figure Lengend Snippet: EZH2 inhibitors EPZ6438 and GSK126 induced M0 macrophages to differentiate into the M1 phenotype. (A) Effects of GSK126 and EPZ6438 on the proliferation in RAW264.7 cells every 4 h until the end of 48 h. (B) Cellular viability of RAW264.7 cells was detected after EPZ6438 and GSK126 treatment. (C) Schematic illustration of EZH2 inhibitor treatment schedule. (D, E) RAW264.7 cells were treated with EPZ-6438 (10, 20 μM) (D) and GSK126 (5, 10 μM) (E) for 48 h. Control cells were maintained in a medium supplemented with DMSO throughout the entire experimental period. An additional group of cells was treated with LPS (100 ng/ml) for the last 12 h. M1-type macrophage genes ( CD86, TNFα, and iNOS ) were analyzed by RT-PCR. (F) Schematic illustration of EZH2 inhibitor treatment schedule. EZH2 inhibitor and LPS (100 ng/ml) or IL-4 (20 ng/ml) were added into the culture medium of RAW264.7 cells at the same time. Effects of EPZ6438 and GSK126 on macrophage polarization were detected at time points of 0.5 h, 1 h, and 2 h, respectively. (G, H) M1-type macrophage genes ( CD86 and iNOS ) (G) and M2-type macrophage genes ( CD206 and Arg1 ) (H) were analyzed by RT-PCR. Data are shown as mean ± SEM and three independent experiments were involved. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance.

Article Snippet: Five-micrometer formalin-fixed, paraffin-embedded (FFPE) tumor sections of the mouse model were stained using a reference protocol ( ) and the panel contained antibodies against DAPI (Cat # : 44010A, BestBio), CD206 (1:500, dye 620, Cat # : 24595, Cell Signaling Technology), CD86 (1:500, dye 690, Cat # : 19589, Cell Signaling Technology), and F4/80 (1:500, dye 540, Cat # :70076, Cell Signaling Technology).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Pharmacological inhibition of host pathways enhances macrophage killing of intracellular bacterial pathogens

doi: 10.1101/2025.04.06.647500

Figure Lengend Snippet: Uninfected (control), 15 µg/ml polyP treated, or Mtb infected macrophages with GM-CSF in the absence (No drug) or presence of 10 µg/ml ScPPX or 1000 nM of the indicated inhibitor at 24 hours after phagocytosis (as described in the bacterial survival assay) were fixed, permeabilized and stained with antibodies against CD54 or CD206, and fluorescence intensities were measured and average of control (B) or No drug (D) was considered 100%. Bars are 100 µm. Fluorescence images are representative of six independent experiments (three female and three male). All values are mean ± SEM of six (three females and three males) independent experiments. One or more than one images from each independent experiment were analyzed, indicated by multiple data points. Male data points are shown in blue and female data points are shown in red. An unpaired t-test was performed to assess male vs. female differences for each bar in the graph. * p < 0.05; ** or ## p < 0.01; **** p < 0.0001 (Kruskal-Wallis test). *indicates compared to no drug; # indicates compared to control. $ p < 0.05 indicates female vs male (unpaired t-test).

Article Snippet: 200 µl of 1:2000 rabbit anti- Mtb antibody (Cat# OBT0947; Bio-Rad), 1:1000 rabbit anti-CD54 (Cat#67836; Cell Signaling, Danvers, MA), 1:1000 rabbit anti-CD206 (Cat#91992; Cell Signaling) antibody in PBS/ 0.1 % Tween 20 (Fisher Scientific, Pittsburgh, PA) (PBST) was added to macrophages and incubated at 4 °C overnight.

Techniques: Control, Infection, Clonogenic Cell Survival Assay, Staining, Fluorescence

Journal: Cancer biology & therapy

Article Title: circ_C20orf11 enhances DDP resistance by inhibiting miR-527/YWHAZ through the promotion of extracellular vesicle-mediated macrophage M2 polarization in ovarian cancer.

doi: 10.1080/15384047.2021.1959792

Figure Lengend Snippet: Figure 5. EVs mediate M2-type macrophage polarization in ovarian cancer. (a) EVs were isolated from the supernatant of SKOV3/DDP cells and characterized via electron microscopy and (b) nanoparticle tracking. (C) EV-specific markers (CD9, TSG101 and CD63) were measured via western blotting. (d) EVs were labeled with PKH67 dye and cultured with THP-1 cells. Representative fluorescence images are shown. (e) After SKOV3/DDP and A2780/DDP cells were transfected with adenoviral circ_C20orf11 (si- SKOV3/DDP-Exo+si-C20orf11) or its vector control (si-SKOV3/DDP-Exo+si-NC), the cells were cultured with EVs from SKOV3/DDP cells. IL-10 expression was detected using an ELISA. Untreated THP-1 cells served as the control. (f) An ELISA was performed to detect IL-6 expression. (g) Relative mRNA expression of TNF-α, IL-6 and iNOS. (H) mRNA expression of CD206, IL-10 and Arg-1 in relation to GAPDH and U6 expression. n = 3. *P < .05, ** P < .01, *** P < .001.

Article Snippet: After centrifugation (1,000 rpm for 8 min at 4°C), the isolated cells were stained using a CD206 antibody (cat. no. 18704-1-AP, Proteintech) according to the manufacturer’s instructions.

Techniques: Isolation, Electron Microscopy, Western Blot, Labeling, Cell Culture, Fluorescence, Transfection, Plasmid Preparation, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cancer biology & therapy

Article Title: circ_C20orf11 enhances DDP resistance by inhibiting miR-527/YWHAZ through the promotion of extracellular vesicle-mediated macrophage M2 polarization in ovarian cancer.

doi: 10.1080/15384047.2021.1959792

Figure Lengend Snippet: Figure 6. Silencing of circ_C20orf11 enhances sensitivity to DDP in vivo. Xenotransplantation studies were performed with SKOV3 cells. (a) Tumor volume was measured every 5 days for 30 days. (b) Representative images of tumor formation. (c) Tumor weight was measured. (d) qPCR analysis of C20orf11, miR-527 and YWHAZ expression. (e) qPCR results showing CD206, IL-10 and Arg-1 expression. (f) Western blotting detection of YWHAZ PD-L1 is presented. n = 3. *P < .05, ** P < .01, *** P < .001.

Article Snippet: After centrifugation (1,000 rpm for 8 min at 4°C), the isolated cells were stained using a CD206 antibody (cat. no. 18704-1-AP, Proteintech) according to the manufacturer’s instructions.

Techniques: In Vivo, Expressing, Western Blot

Journal: Cancer biology & therapy

Article Title: circ_C20orf11 enhances DDP resistance by inhibiting miR-527/YWHAZ through the promotion of extracellular vesicle-mediated macrophage M2 polarization in ovarian cancer.

doi: 10.1080/15384047.2021.1959792

Figure Lengend Snippet: Figure 7. Serum EV-circ_C20orf11 levels are upregulated in ovarian patients. Patients were considered DDP resistant if they showed no significant clinical effect or had progressive disease after receiving one cycle of DDP treatment. The remaining patients were considered DDP sensitive. Real-time qPCR analysis of (a) C20orf11 and (b) miR-527 expression in relation to GAPDH and U6 expression in DDP-sensitive ovarian cancer tissue (S), DDP-resistant ovarian cancer tissue (R), and healthy ovarian tissue (Normal). (c) Flow cytometry detection and quantification of CD206-positive cells in DDP-sensitive ovarian cancer tissue (S), DDP-resistant ovarian cancer tissue (R), and healthy ovarian tissue (Normal). (d) Real-time qPCR analysis of CD206 expression in relation to GAPDH and U6 expression in DDP-sensitive ovarian cancer tissue (S), DDP- resistant ovarian cancer tissue (R), and healthy ovarian tissue (Normal). (e) The expression level of C20orf11 in ovarian cancer tissue was negatively correlated with that of miR-527. (f) The expression level of miR-527 in ovarian cancer tissue was negatively correlated with that of CD206. (g) Representative image of serum EVs detected using an electron microscope. (h) EVs were measured using nanoparticle tracking. (i) EV markers were analyzed via western blotting. (j) The abundance of C20orf11 in serum EVs was assessed using qPCR. (k) Kaplan-Meier survival curves of patients with ovarian cancer with high and low C20orf11 expression. n = 3. *P < .05, ** P < .01.

Article Snippet: After centrifugation (1,000 rpm for 8 min at 4°C), the isolated cells were stained using a CD206 antibody (cat. no. 18704-1-AP, Proteintech) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Microscopy, Western Blot

Journal: Cell stress & chaperones

Article Title: COL5A2-mediated endoplasmic reticulum stress promotes macrophage M2 polarization in lung adenocarcinoma.

doi: 10.1016/j.cstres.2025.100081

Figure Lengend Snippet: Fig. 2 COL5A2 mediates ER stress to promote macrophage M2 polarization. (a) GSEA analysis of the signaling pathways enriched by differential expression of COL5A2; (b) CCK-8 assay detected cell viability in various groups; (c) WB assessed the protein expression levels of ER stress-related proteins (GRP78 and CHOP) in cells of various groups; (d) IF of the expression of M2 macrophage marker (CD206) in various groups, scale bar = 100 µm; (e) flow cytometry analysis of the proportion of CD68 and CD206 double-positive macrophages in various groups; (f) qPCR analysis of the mRNA expression levels of TGF-β and IL-10 in various groups. *P < 0.05. Abbreviations used: CCK-8, cell counting kit-8ER; endoplasmic reticulum; IF, immunofluorescence; qPCR, quantitative polymerase chain reaction; WB, Western blot.

Article Snippet: For cellular assays, cells were seeded in 6-well plates, fixed with 4% paraformaldehyde for 20 min, washed, permeabilized with 0.5% Triton-X 100 for 30 min, blocked, and incubated with a CD206 antibody (59414, Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Protein-Protein interactions, Quantitative Proteomics, CCK-8 Assay, Expressing, Marker, Flow Cytometry, Cell Counting, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell stress & chaperones

Article Title: COL5A2-mediated endoplasmic reticulum stress promotes macrophage M2 polarization in lung adenocarcinoma.

doi: 10.1016/j.cstres.2025.100081

Figure Lengend Snippet: Fig. 4 COL5A2-mediated ER stress amplifies PD-L1-rich exosome release to enhance macrophage M2 polarization. (a) WB analysis of PD-L1 protein levels in exosomes among different groups; (b) PKH26 staining assessed the uptake of exosomes by macrophages, scale bar = 200 µm; (c) WB analysis of PD-L1 protein levels in macrophages among different groups after exosome uptake by macrophages; (d) IF analyzed the expression of M2 macrophage marker (CD206) among different groups, scale bar = 100 µm; (e) flow cytometry determined the ratio of CD68+ and CD206+ double-positive macrophages among different groups; (f) qPCR measured mRNA levels of TGF-β and IL-10 among different groups. *P < 0.05. Abbreviations used: ER, endoplasmic reticulum; IF, immunofluorescence; qPCR, quantitative polymerase chain reaction; WB, Western blot.

Article Snippet: For cellular assays, cells were seeded in 6-well plates, fixed with 4% paraformaldehyde for 20 min, washed, permeabilized with 0.5% Triton-X 100 for 30 min, blocked, and incubated with a CD206 antibody (59414, Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Staining, Expressing, Marker, Flow Cytometry, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell stress & chaperones

Article Title: COL5A2-mediated endoplasmic reticulum stress promotes macrophage M2 polarization in lung adenocarcinoma.

doi: 10.1016/j.cstres.2025.100081

Figure Lengend Snippet: Fig. 5 In vivo impact of COL5A2 on macrophage M2 polarization. (a) and (b) Tracking tumor growth and tumor size; (c) IHC evaluation of cell proliferation marker KI-67, scale bar = 50 µm; (d) WB profiling of COL5A2 and PD-L1 proteins; (e) Immunofluorescence for M2 macrophage marker CD206 expression, scale bar = 50 µm; (f) flow cytometry analysis of CD68+CD206+ macrophage populations; (g) WB detection of ER stress indicators GRP78 and CHOP. *P < 0.05. Abbreviations used: ER, endoplasmic reticulum; IHC, immunohistochemistry.

Article Snippet: For cellular assays, cells were seeded in 6-well plates, fixed with 4% paraformaldehyde for 20 min, washed, permeabilized with 0.5% Triton-X 100 for 30 min, blocked, and incubated with a CD206 antibody (59414, Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: In Vivo, Marker, Immunofluorescence, Expressing, Flow Cytometry, Immunohistochemistry

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASC therapeutic effects on experimental periodontitis in rats. (A) The schematic diagram showed the induction of the experimental rat model of periodontitis for 21 days and ASC injection after the removal of ligatures. (B) Micro-CT imaging and bone parameters (CEJ-ABC distance, BV/TV, Tb. Th, and Tb. Sp) between healthy and experimental rats of periodontitis. Scale bar, 1 mm. (C) Micro-CT imaging displayed the alveolar bone of PBS-injected and ASC-injected rats on day 7, day 14, and day 21 Scale bar, 1 mm. (D) The CEJ-ABC distance and bone parameters (BV/TV, Tb. Th, and Tb. Sp) between the PBS-injected and ASC-injected rats. (E) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (F) The calculated ratio of iNOS + /CD206 + macrophages in PBS-injected and ASC-injected groups. CEJ-ABC, cementoenamel junction and alveolar bone crest. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Injection, Micro-CT, Imaging, Immunofluorescence, Staining

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASCs modulated macrophage polarization through NRF2. (A) Representative immunohistochemical staining of NRF2 in the PBS-injected and ASC-injected rats on day 7, day 14, and day 21. Scale bar, 50 μm. (B) Representative immunofluorescence staining of NRF2 (Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (C) Schematic diagram of knocking down NRF2 in macrophages and macrophages cocultured with ASCs and LPS stimulation. (D, E) RT-qPCR and western blotting were used to measure the NRF2 siRNA or siNC in macrophages. (F, G) The mRNA expression and protein levels of M1 markers were increased, while those of M2 markers were decreased after silencing NRF2 in the cocultured groups. R, root; PDL, periodontal ligament; AB, alveolar bone; siRNA, small interfering RNA; NC, negative control. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Injection, Immunofluorescence, Quantitative RT-PCR, Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: Inhibition of IDO activity reduced the therapeutic potential of ASCs in experimental periodontitis and downregulated NRF2 expression. (A) Three-dimensional reconstruction of maxillary alveolar bone in ASC-injected and 1-MT pretreated ASC-injected rats on day 7, day 14, and day 21. Scale bar, 1 mm. (B) Analysis of bone parameters. (C) Immunohistochemical staining of IL-1β, OCN, and NRF2 in the ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 50 μm. (D) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 20 μm. (E) The mean IOD of IL-1β, OCN, and NRF2 and the calculated ratio of iNOS + /CD206 + . IOD, integrated optical density.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Expressing, Injection, Immunohistochemical staining, Staining, Immunofluorescence