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: Aging Cell

Article Title: Telomerase Knockout in Myeloid Cells Predisposes Mice to Foam Cell Formation, Dyslipidemia, Lung Fibrosis, and Cardiac Dysfunction

doi: 10.1111/acel.70490

Figure Lengend Snippet: Tert KO induces lipid‐associated macrophages (LAMs). Starch‐induced i.p. macrophages from 2‐year‐old female mice were analyzed. (a) Upon adherence in primary culture, IF with antibodies against CD80 (M1‐macrophage) and CD206 (M2‐polarization) markers reveals a lower frequency of CD206 + macrophages in KO mice. (b) Data quantification from multiple fields of view in (a), indicating macrophage polarization shift toward the M1 phenotype. (c) Upon LPS (100 ng/mL, 4 h) treatment in primary culture, q‐RT‐PCR (normalized to 18S RNA) demonstrates higher expression of genes coding for inflammation markers IL1 and IL6 in KO macrophages. (d) Macrophages were induced to convert into foam cells by oxLDL (0.025 mg/mL) treatment for 24 h. Note increased uptake of red‐fluorescent C 12 ‐BODIPY (0.3 μM, 5 min) by mG+ KO cells (yellow arrows) compared to mG+ WT cells (green arrows) in primary culture. (e) q‐RT‐PCR (normalized to 18S RNA) demonstrates lower expression of genes coding for lipid efflux effectors APOE, LDLR, ABCA1, ABCG1, and higher expression of genes coding for lipid transporters CD36 and FABP5 in KO oxLDL‐treated macrophages. (f) OxLDL‐treated macrophages stained with Oil Red O: Note larger lipid droplets (arrows) in KO cells. For all data, mean+/− SEM (error bars). * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐sided Student's t ‐test). Scale bar: 50 μm.

Article Snippet: For immunofluorescence (IF), the following antibodies were used: CD206 (R&D Systems, AF2534, 1:75); CD80 (Abclonal A23688, 1:50); CD68 (Invitrogen, MA5‐1324, 1:75); F4/80 (Abcam, ab16911, 1:50); TERT (Biossusa, bs‐1411R, 1:50).

Techniques: Starch, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining

Journal: Aging Cell

Article Title: Telomerase Knockout in Myeloid Cells Predisposes Mice to Foam Cell Formation, Dyslipidemia, Lung Fibrosis, and Cardiac Dysfunction

doi: 10.1111/acel.70490

Figure Lengend Snippet: AT abnormalities in LysM‐ Tert KO mice. (a) Senescence‐associated β‐galactosidase staining of VAT from 20‐month‐old female mice. (b) Senescence‐associated β‐galactosidase staining of adherent cells from VAT in (a). Arrows: Senescent cells. (c) Flow cytometry on VAT from A, revealing a lower frequency of mG+ macrophages expressing CD206 in KO mice. (d) Flow cytometry on SAT, revealing a higher frequency of mG+ macrophages expressing CD86 in KO mice. (e) IF with antibodies against CD68 and CD206 reveals a lower frequency of CD206+ macrophages (red arrows) in SAT of KO mice. IF with antibodies against perilipin‐1 and F4/80 reveals comparable adipocyte size in SAT of WT and KO mice. (f) Trichrome staining reveals fibrosis (arrows) in SAT of KO mice. In (d, e) 4‐month‐old male mice fed an atherogenic diet were used. Scale bar: 50 μm.

Article Snippet: For immunofluorescence (IF), the following antibodies were used: CD206 (R&D Systems, AF2534, 1:75); CD80 (Abclonal A23688, 1:50); CD68 (Invitrogen, MA5‐1324, 1:75); F4/80 (Abcam, ab16911, 1:50); TERT (Biossusa, bs‐1411R, 1:50).

Techniques: Staining, Flow Cytometry, Expressing

Journal: Aging Cell

Article Title: Telomerase Knockout in Myeloid Cells Predisposes Mice to Foam Cell Formation, Dyslipidemia, Lung Fibrosis, and Cardiac Dysfunction

doi: 10.1111/acel.70490

Figure Lengend Snippet: Lung abnormalities in LysM‐ Tert KO mice. (a) IF with antibodies against F4/80 and CD206 reveals a lower frequency of CD206+ macrophages (red arrows) in lungs of 6 month‐old chow‐fed KO mice, compared to WT mice. (b) Data quantification for 10 view fields from A. * p < 0.05 (two‐sided Student's t ‐test). (c) Trichrome staining reveals fibrosis (arrows) in 20‐month‐old lungs of KO mice. (d) q‐RT‐PCR (normalized to 18S RNA) demonstrates higher expression of Tgfb1 and Cola1a in lungs of KO male and female mice. Shown are mean+/− SEM (error bars). * p < 0.0001 (two‐sided Student's t ‐test). ** p < 0.01. In (a), (b), and (d), 6‐month‐old male mice fed a chow diet were used. Scale bar: 50 μm.

Article Snippet: For immunofluorescence (IF), the following antibodies were used: CD206 (R&D Systems, AF2534, 1:75); CD80 (Abclonal A23688, 1:50); CD68 (Invitrogen, MA5‐1324, 1:75); F4/80 (Abcam, ab16911, 1:50); TERT (Biossusa, bs‐1411R, 1:50).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides

doi: 10.1371/journal.pone.0160602

Figure Lengend Snippet: Liver sections were fixed with cold acetone for 10 min, washed in PBS and incubated overnight at 4°C with polyclonal rabbit anti-FPR1 (1:200), followed by Alexa Fluor ® -488 goat anti-rabbit (1:1000). Intense FPR1 staining can be observed along the sinusoids in mouse liver (A), while in human liver the staining was more evenly localized to both LSECs and Heps (B). Double staining was performed using polyclonal rabbit anti-FPR1, rat anti-mouse CD206, and goat anti-human CD206, followed by Alexa Fluor ® -488 and Alexa Fluor ® -594 secondary antibodies, respectively.

Article Snippet: Polyclonal rabbit anti-FPR1 (H-230-Santa Cruz Biotech), rat-anti mouse CD206, clone MCA2235 (AbD Serotec, Oxford, UK), goat anti-human CD206 (R&D Systems), Alexa Fluor ® -488 and -594 conjugated secondary antibodies (InVitrogen), and Draq5 (BioStatus Limited, Leicestershire, UK) were used for immunolabeling of cells and tissue.

Techniques: Incubation, Staining, Double Staining

Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: TP-0903 treatment suppresses the population of CD206 + macrophages in human IBC xenograft and murine TNBC syngeneic models. A Tumors from a SUM149 xenograft mouse model treated with vehicle or TP-0903 for 7 days were dissociated to obtain a single-cell suspension and stained with antibodies. Flow cytometric analysis showed a decreased CD206 + macrophage population (CD45 + CD11b + Ly6C − Ly6G − F4/80 + CD206 + cells) after TP-0903 treatment. B IHC staining of CD206 on slides from the above tumor sections. TP-0903 decreased the population of CD206 + cells in SUM149 tumor tissues. Left panel: representative IHC staining images. Scale bar = 200 μm. Right panel: quantification of CD206 expression by ImageJ. C Tumor growth curves for the vehicle- and TP-0903–treated groups in murine TNBC syngeneic mouse models. TP-0903 suppressed the growth of 4T1.2 and E0771 mammary tumors in vivo. D TP-0903 treatment reduced CD206 + macrophages in murine mammary tumors from the 4T1.2 mouse model. E IHC staining for CD206 showed reduced CD206 + cells in TP-0903–treated tumor tissues from E0771 mice. Left panel: representative IHC staining images. Scale bar = 200 μm. Right panel: quantification of CD206 expression. F Flow cytometric analysis showed a decrease in Tregs (CD45 + CD3 + CD4 + CD25 + FOXP3 + cells) in TP-0903–treated 4T1.2 and E0771 mice. 4T1.2 syngeneic model: n = 10 mice; E0771 syngeneic model: n = 15 mice. Data were summarized as means ± SD in A , B , and D to F and means ± SEM in C . A 2-tailed Student t test was used to calculate P values. * P < 0.05; ** P < 0.01

Article Snippet: The following primary antibodies were used for Western blotting: anti-human phospho-AXL ([Y779] R&D Systems, Minneapolis, MN; AF2228), anti-human AXL (Sigma; WH0000558M1), anti-mouse AXL (R&D Systems; AF854), anti-human phospho-STAT6 ([Tyr641] Cell Signaling Technology, Danvers, MA; 56554S), anti-human STAT6 (Cell Signaling Technology; 9362S), anti-human CD206 (R&D Systems; MAB25341), and anti-human β-Actin (Sigma; A5316).

Techniques: Suspension, Staining, Immunohistochemistry, Expressing, In Vivo

Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: AXL inhibition suppresses the polarization of immunosuppressive M2 macrophages. A AXL was knocked out in human THP-1 monocytes using the CRISPR/Cas9 system. These cells and control cells were subsequently induced into M2 macrophages, and AXL depletion was confirmed in AXL-KO M2 macrophages using Western blotting. M2 macrophages polarized from AXL-KO THP-1 cells had lower expression of AXL than M2 macrophages polarized from control cells. B The expression of M2 macrophage markers and secreted cytokines was measured by qRT-PCR. AXL KO reduced the expression of CD163 , CD206 , CCL17 , and CCL18 in M2 macrophages. C Flow cytometric analysis was conducted to compare the CD206 + macrophage population polarized from AXL-KO versus control THP-1 cells. AXL KO reduced the population of CD206 + macrophages polarized from THP-1 cells. D Treatment with TP-0903 inhibited the expression of AXL mRNA (left panel) and protein (right panel) in THP-1–derived M2 macrophages as determined using qRT-PCR and Western blotting, respectively. E TP-0903–treated M2 macrophages showed reduced mRNA level of CD163 , CD206 , CCL17 , and CCL18 compared to vehicle-treated M2 macrophages. F Flow cytometry and Western blotting showed that TP-0903 reduced the population of CD206 + cells (left panel) and CD206 protein expression (right panel) in THP-1–derived M2 macrophages. All experiments were repeated at least three times. Data were summarized as means ± SD. A 2-tailed Student t test ( B and 1-way analysis of variance followed by Dunnett’s multiple comparison test ( D – F ) were used to calculate P values. * P < 0.01

Article Snippet: The following primary antibodies were used for Western blotting: anti-human phospho-AXL ([Y779] R&D Systems, Minneapolis, MN; AF2228), anti-human AXL (Sigma; WH0000558M1), anti-mouse AXL (R&D Systems; AF854), anti-human phospho-STAT6 ([Tyr641] Cell Signaling Technology, Danvers, MA; 56554S), anti-human STAT6 (Cell Signaling Technology; 9362S), anti-human CD206 (R&D Systems; MAB25341), and anti-human β-Actin (Sigma; A5316).

Techniques: Inhibition, CRISPR, Control, Western Blot, Expressing, Quantitative RT-PCR, Derivative Assay, Flow Cytometry, Comparison

Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: AXL suppression inhibits the polarization of immunosuppressive M2 macrophages via STAT6. A Treatment with TP-0903 reduced AXL, phospho-STAT6, and STAT6 protein expression as determined by Western blotting. B M2 macrophages polarized from AXL-KO THP-1 cells had lower phospho-AXL, AXL, phospho-STAT6, and STAT6 protein expression than those polarized from control THP-1 cells, as determined using Western blotting. C–E STAT6 was knocked down in THP-1 cells using siRNAs, and then THP-1 cells were induced to M2 macrophages. Knockdown of STAT6 in THP-1–polarized M2 macrophages C decreased the CD163 + CD206 + macrophage population as determined by flow cytometry D and decreased the expression of the CD163 and CD206 genes as determined using qRT-PCR E . F STAT6 was overexpressed in M2 macrophages polarized from AXL-KO THP-1 cells as tested using qRT-PCR. G STAT6 overexpression mitigated the inhibitory effect of AXL KO on the expression of M2 macrophage markers and cytokines, including CD163 , CD206 , CCL17 , and CCL18 . H The CM from control, AXL-KO, and AXL-KO + STAT6–overexpressing M2 macrophages and fresh media were used as attractants plated in the bottom chamber of transwells to test the migration of SUM149 cells. Migration of SUM149 cells was greater with CM from AXL-KO + STAT6–overexpressing M2 macrophages than with CM from AXL-KO M2 macrophages. All experiments were repeated at least three times. Data were summarized as means ± SD. One-way analysis of variance followed by Dunnett’s multiple comparison test was used to calculate P values. * P < 0.05; ** P < 0.01

Article Snippet: The following primary antibodies were used for Western blotting: anti-human phospho-AXL ([Y779] R&D Systems, Minneapolis, MN; AF2228), anti-human AXL (Sigma; WH0000558M1), anti-mouse AXL (R&D Systems; AF854), anti-human phospho-STAT6 ([Tyr641] Cell Signaling Technology, Danvers, MA; 56554S), anti-human STAT6 (Cell Signaling Technology; 9362S), anti-human CD206 (R&D Systems; MAB25341), and anti-human β-Actin (Sigma; A5316).

Techniques: Expressing, Western Blot, Control, Knockdown, Flow Cytometry, Quantitative RT-PCR, Over Expression, Migration, Comparison

Journal: Carbohydrate polymers

Article Title: The immunogenicity of p24 protein from HIV-1 virus is strongly supported and modulated by coupling with liposomes and mannan.

doi: 10.1016/j.carbpol.2024.121844

Figure Lengend Snippet: Fig. 2. Interaction of p24 protein and mannan with liposomes. A) The scheme of the specific immunogold staining of p24 and mannan molecules. Anti-V5 antibody was used for detection of p24 antigen, whereas His-tagged recombinant mannose receptor and anti-HisTag antibody were used for the detection of mannan molecules on the surface of liposomes. B) Analysis of p24 binding on metallochelating liposomes, or C) p24 binding on mannosylated liposomes using isothermal titration. In the negative controls experiments, where no reaction enthalpy is observed, the results are shown as non-binding. Thermodynamic parameters of interaction between p24 antigen and metallochelation liposomes or mannosylated liposomes are shown in the inserted table. D) Overview image of p24 protein on the surface of liposomes visualized by anti-V5 antibody detected by protein A gold-nanoparticles. E) Detail of single liposome with immunogold stained surface p24 protein. F) p24 mannosylated liposome stained with MMR (His tagged recombinant human mannose receptor CD206) – anti-HisTag antibody – Protein A gold nanoparticles. G) Control non-stained p24 mannosylated liposomes. H) Control - p24 mannosylated liposome stained with Protein A gold nanoparticles, without MMR – HisTag antibody. I) Control - p24 mannosylated liposome stained with MMR – Protein A gold nanoparticles, without HisTag antibody.

Article Snippet: Briefly, 4 μg of recombinant human mannose receptor CD206 (MMR, R&D Systems; Minneapolis, USA) was mixed with 3 mg of mannosylated liposomes (10 mg/ml PBS).

Techniques: Liposomes, Staining, Recombinant, Binding Assay, Titration, Control

Journal: Journal of Clinical Laboratory Analysis

Article Title: High‐dimensional single‐cell proteomics analysis reveals the landscape of immune cells and stem‐like cells in renal tumors

doi: 10.1002/jcla.23155

Figure Lengend Snippet: Purified antibodies about the immune cell‐centric panel

Article Snippet: CD206/MMR , 168Er , 43511 , Fluidigm , 3168008B.

Techniques: Purification