Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1 and TNF- α protein expression in 33.3 mM glucose-induced macrophage. (a) iNOS and CD206 protein expressions. (d) Arg-1 protein expression. (f) TNF- α protein expression. The results of iNOS, CD206, Arg-1, and TNF- α were represented in (b), (c), (e), and (g), respectively. All results were expressed as a ration with respect to control and represented as the mean ± SD in triplicates. ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing, Control

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1, and TNF- α mRNA expressions in 33.3 mM glucose-induced macrophage. (a) iNOS mRNA expression. (b) CD206 mRNA expression. (c) CD206 mRNA expression. (d) TNF- α mRNA expression.All the results were represented as the mean ± SD in triplicates ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors

doi: 10.3389/fimmu.2020.01412

Figure Lengend Snippet: Polarization of M0 and M2 macrophages after treatment with free and nanocomplexed poly(I:C). Expression of the M2 markers (A–D) CD206 and (E–H) CD163 in ENCP-treated M0 and M2 macrophages, in comparison to the prototypic phenotypes evaluated by FACS. M2 M1 represents M2 macrophages that were treated with LPS + IFN-γ for their M1 polarization. Macrophages were incubated with the treatments for 48 h, and the poly(I:C) dose used was 5 μg/mL. Each symbol shape represents a different donor. Values are shown as mean ± SD ( n ≥ 3). Statistical comparison was done using an ordinary one-way ANOVA followed by a Tukey's comparison test between groups; or a paired t -test for (C,G) . Statistically significant differences are represented as ** p < 0.01. C12r8, laurate-octaarginine; HA, hyaluronic acid; pArg, poly-arginine; PEG–PGA, pegylated polyglutamic acid; pIC, poly(I:C).

Article Snippet: They were then stained with APC-mouse anti-human HLA-DR (552764, BD Biosciences, CA, USA), FITC-mouse anti-human mannose receptor CD206 (551135), anti-human CD163-BV421 (562643), CD80 APC-H7-mouse anti-Human CD80 (Clone L307.4; 561134) and anti-human CD68-PE (556078) (all from BD Biosciences, CA, USA).

Techniques: Expressing, Incubation

Journal: Frontiers in Immunology

Article Title: Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors

doi: 10.3389/fimmu.2020.01412

Figure Lengend Snippet: Schematic illustration of the in vitro effects of the poly(I:C) ENCPs developed in this study. (A) Upon interaction with macrophages, poly(I:C) nanocomplexes are taken up. (B) This process allows poly(I:C) to reach its target receptor TLR3, found in the endosomes. It is expected that this interaction activates the TLR3 and the TRIF pathway, stimulating the upregulation of type I IFN genes. (C) The expression of M2 (CD206 and CD163) surface markers was slightly decreased, while M1 (CD80 and MHC II) markers are not substantially modified. Nevertheless, (D) CXCL10 and CCL5 chemokines, involved in the attraction of CD8 T cells to the tumor microenvironment, are secreted. (E) The direct cytotoxicity of macrophages toward cancer cells is also enhanced. Images were reproduced from Servier Medical Art under a Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licenses/by/3.0 .

Article Snippet: They were then stained with APC-mouse anti-human HLA-DR (552764, BD Biosciences, CA, USA), FITC-mouse anti-human mannose receptor CD206 (551135), anti-human CD163-BV421 (562643), CD80 APC-H7-mouse anti-Human CD80 (Clone L307.4; 561134) and anti-human CD68-PE (556078) (all from BD Biosciences, CA, USA).

Techniques: In Vitro, Expressing, Modification

Journal: Biochemical and biophysical research communications

Article Title: miR-210 promotes hepatocellular carcinoma progression by modulating macrophage autophagy through PI3K/AKT/mTOR signaling.

doi: 10.1016/j.bbrc.2023.04.055

Figure Lengend Snippet: Fig. 1. Differentiation of human THP-1 monocytes to M2 macrophages. *P < 0.05, **P < 0.01, ***P < 0.001. (A) Morphology of THP-1 monocytes, M0 macrophages (non-polarized THP-1 cells), and M2 polarized macrophages. (B) Flow cytometry showed cell surface expression of CD11b, CD68 and CD206 in the THP-1, M0, M2 populations. (C) The levels of expression of M2 macrophage markers mRNA and miR-210 were examined by qRT-PCR. All results showed relative fold changes relative to M0 (non-polarized THP-1) cells. (D) Detection of Arg-1 protein expression in M2 macrophages by Western blot.

Article Snippet: Flow cytometry antibodies included: FITC anti-human CD68 (321103, BioLegend), PE anti-human CD11b (301305, BioLegend), FITC anti-human CD206 (Sc-20060FITC, Santa cruz).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Nature Communications

Article Title: IL-33 contributes to sepsis-induced long-term immunosuppression by expanding the regulatory T cell population

doi: 10.1038/ncomms14919

Figure Lengend Snippet: ( a – e ) Lung tissue and peritoneal cells from C57BL/6 J ( a , c , d ) and BALB/c ( b , e ) mice undergoing CLP and antibiotic treatment were harvested at the indicated time points. ( a ) mRNA expression of Cebpb (C/EBPβ) , Arg1 (arginase-1), Mrc1 (MR) and Igf1 in the total lungs were determined by RT-qPCR at indicated times after CLP ( n ≥4 mice per group). ( b ) Lungs obtained from six mice, either naive or CLP (day 10 after CLP), were pooled from two independent samples and CD11b + cells were isolated. mRNA expression of Arg1 (arginase-1), Mrc1 (MR), Rentla (encoding Fizz1) in isolated CD11b + cells were determined by RT-qPCR. ( c ) IGF-1, CCL17 and CCL22 concentrations in the lungs were determined by ELISA at indicated times after CLP ( n ≥3 mice per group). ( d ) Representative FACS plots and frequency of peritoneal CD206 + F4/80 + macrophages at indicated times after CLP ( n ≥3 mice per group). ( e ) Representative western blot of Arg1 (Arginase-1) expression in the peritoneal cells at day 10 after CLP ( n =9 for naive group and n =5 for CLP group). ND, not detected. * P <0.05, ** P <0.01 and *** P <0.001 (one-way ANOVA result with Dunnett posthoc tests in a , c , two-tailed unpaired Student's t -test in b , d ). Data are from one ( b ) and representative of two ( a , c – e ) independent experiments (mean±s.e.m. in a – d ).

Article Snippet: To stain for surface antigens, cells were incubated with specific antibodies to F4/80 (BM8, eBioscience), CD206 (mannose receptor C type 1, MR; MR5D3, AbD Serotec), CD4 (GK1.5, eBioscience; H129.19, BD Biosciences), CD4 (RPA-T4, BD Biosciences, for human), CD45 (30-F11, BD Biosciences), Ly-6A/E (Sca-1; E13-161.7, BioLegend), CD3e (145-2C11, BD Biosciences), CD8a (53-6.7, eBioscience), CD19 (eBIO1D3, 1D3, eBioscience; 6D5, BioLegend), CD11b (M1/70, BioLegend), CD11c (N418, BioLegend; Hl3, BD Biosciences), CD49b/Pan-NK (DX5, BD Biosciences), FcɛRI Alpha (MAR-1, eBioscience), T1/St2 (IL-33R, DJ8, MD Biosciences) or the appropriate isotype controls for 30 min. To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with mouse Foxp3 Buffer Set (BD Biosciences), according to the manufacturer's recommendations.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: IL-33 contributes to sepsis-induced long-term immunosuppression by expanding the regulatory T cell population

doi: 10.1038/ncomms14919

Figure Lengend Snippet: ( a ) Peritoneal cells were collected from C57BL/6 J or Rag1 −/− mice 15 days after CLP and antibiotic treatment and the frequency of F4/80 + CD206 + macrophages determined by FACS ( n ≥3 mice per group). ( b ) Peritoneal cells of BALB/c, Il1rl1 −/− and Stat6 −/− mice were harvested in ‘ a ' above and the frequency of F4/80 + CD206 + macrophages determined by FACS ( n ≥5 mice per group). ( c ) Representative FACS plots of IL-4Rα + F4/80 + peritoneal macrophages of C57BL/6 J mice at day 15 after CLP ( n =5 per group). ( d , e ) Peritoneal macrophages harvested in b above were stimulated with IL-4 in vitro . ( d ) STAT6 phosphorylation in the cells was determined by FACS ( n =4 per group). ( e ) Concentrations of CCL22 in the 18 h culture supernatants determined by ELISA ( n =4 mice per group). ( f ) Peritoneal cells of BALB/c, Il1rl1 −/− and Stat6 −/− mice were harvested in a above. The number of viable bacteria recovered from lysates of peritoneal macrophages exposed, in vitro , to L. pneumophila for 72 h in the presence or absence of arginase inhibitor BEC. ( g ) BALB/c macrophages from BMDM were cultured for 2 days with M-CSF (M0) or IL-4, IL-13 and IL-33 (M2). The number of viable bacteria recovered from lysates of M2-polarized macrophages exposed, in vitro , to L. pneumophila for 72 h in the presence or absence of BEC. ( h ) M0 or M2 macrophages were adoptively transferred (4 × 10 6 cells, i.v.) into naive BALB/c mice, which were challenged 12 days later with L. pneumophila . Survival of mice was recorded ( n =10 mice for M0 group and n =11 for M2 group). ND, not detected. * P <0.05, ** P <0.01 and *** P <0.001 (two-tailed unpaired Student's t- test in a , b , one-way ANOVA result with Bonferroni's posthoc tests in d , Mantel-Cox log-rank test in e ). Data are representative of two ( a , c – g ) independent experiments; or pooled from two ( b , h ) experiments (mean±s.e.m. in a , b , e – h ).

Article Snippet: To stain for surface antigens, cells were incubated with specific antibodies to F4/80 (BM8, eBioscience), CD206 (mannose receptor C type 1, MR; MR5D3, AbD Serotec), CD4 (GK1.5, eBioscience; H129.19, BD Biosciences), CD4 (RPA-T4, BD Biosciences, for human), CD45 (30-F11, BD Biosciences), Ly-6A/E (Sca-1; E13-161.7, BioLegend), CD3e (145-2C11, BD Biosciences), CD8a (53-6.7, eBioscience), CD19 (eBIO1D3, 1D3, eBioscience; 6D5, BioLegend), CD11b (M1/70, BioLegend), CD11c (N418, BioLegend; Hl3, BD Biosciences), CD49b/Pan-NK (DX5, BD Biosciences), FcɛRI Alpha (MAR-1, eBioscience), T1/St2 (IL-33R, DJ8, MD Biosciences) or the appropriate isotype controls for 30 min. To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with mouse Foxp3 Buffer Set (BD Biosciences), according to the manufacturer's recommendations.

Techniques: In Vitro, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Bacteria, Cell Culture, Two Tailed Test