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 Nanobiotechnology

Article Title: A molybdenum oxide-based degradable nanosheet for combined chemo-photothermal therapy to improve tumor immunosuppression and suppress distant tumors and lung metastases

doi: 10.1186/s12951-021-01162-2

Figure Lengend Snippet: Representative fluorescent images ( A ) and a quantitative assay ( B ) of MCF-7 cells treated with FA-BSA-PEG/MoOx@C6, PEG/MoOx@C6 for 1, 2, 4 h; the scale is 100 μm; (* p < 0.05, ** p < 0.01, *** p < 0.001). C Apoptosis results of MCF-7 cells treated with different preparations for 24 h; D quantitative analyses of apoptosis statistics results of MCF-7 cells treated with different formulations. The result of Q2 plus Q3 (n = 3); Flow cytometry analysis of the expression of E CD86 (M1 macrophage marker) and F CD206 (M2 macrophage marker) on macrophages treated by different formulations. G The CLSM images of CRT translocation after treated with different formulations. The green and blue represent CRT and nuclei, respectively. Scale bar: 10 μm; expression of HSP70 ( H ) and HSP70/β—actin ratio ( J ) after different treatments were compared

Article Snippet: 100× Penicillin–Streptomycin–Gentamicin Solution (P1410), PBS, Trypsin–EDTA solution, 0.25% (without phenol red) (9002-07-7), Calcein AM/PI live/dead cell double staining kit (CA1630), 5% BSA Blocking Buffer (SW3015), Percoll Cell separation solution (65455-52-9), Red blood cell lysis buffer (R1010), Collagenase IV (9001-12-1), DNase I (9003-98-9) were purchased from Beijing Solarbio Science & Technology Co., Ltd; Cell counting kit-8 (Catalog No. K1018) was purchased from APExBIO technology Ltd.; DAPI staining solution (C1002) was purchased from Beyotime biotechnology; Annexin V-FITC/PI apoptosis detection kit (40302ES50) was purchased from Shanghai Yisheng biotechnology Co., Ltd.; Fetal bovine serum (11011-8611) was purchased from Zhejiang tianhang biology Co., Ltd; Dulbecco's modified eagle medium (30030), Roswell Park Memorial Institute (RPMI) 1640 medium, CD11b monoclonal antibody (M1/70), APC (17-0112-82), FOXP3 monoclonal antibody (3G3), alexa fluor 647 (MA5-18160), CD11c monoclonal antibody (N418), FITC (11-0114-85), CD80 (B7-1) monoclonal antibody (16-10A1), APC (17-0801-82), CD86 (B7-2) monoclonal antibody (GL1), PE (12-0862-82), CD3 monoclonal antibody (17A2), APC (17-0032-80), CD8a monoclonal antibody (53–6.7), PE (12-0081-85), CD4 monoclonal antibody (GK1.5), FITC (11-0041-81), CD45 monoclonal antibody (30-F11), FITC (11-0451-82), F4/80 monoclonal antibody (BM8), APC (17-4801-80) were purchased from eBioscienceTM; APC anti-mouse CD86 (105011), PE anti-mouse CD206 (MMR) (141705), Recombinant mouse IL-4 (carrier-free) (574302) were purchased from BioLegend; Calreticulin polyclonal antibody (27298-1-AP) was purchased from Proteintech; TNF-α, IFN-γ, IL-6, IL-2 ELISA kits (EK202/2-48) were purchased from Multisciences (Lianke) Biotech, Co., Ltd.

Techniques: Flow Cytometry, Expressing, Marker, Translocation Assay

Journal: iScience

Article Title: Aurkb deficiency disrupts microglial development, homeostasis and hinders remyelination following cuprizone-induced demyelination

doi: 10.1016/j.isci.2026.114718

Figure Lengend Snippet: Aurkb loss transiently elevates CD68 in homeostatic microglia but compromises its upregulation in an LPS-induced inflammation model (A–D) Adult Aurkb fl/fl and Cx3cr1 CreERT2/+ Aurkb fl/fl littermates (8-week-old) were i.p. injected with TAM for 5 consecutive days, followed by tissue collection at 1-month and 3-month post TAM induction. Representative immunofluorescence and quantification of CD68 in microglia at (A and B) 1-month and (C and D) 3-month post-TAM induction ( n = 5 mice per genotype per time point, Scale bars: 50 μm). (E–H) Neonatal Aurkb fl/fl and Cx3cr1 CreERT2/+ Aurkb fl/fl littermates were i.p. injected with TAM for 3 consecutive days at P1-P3, followed by tissue collection at P13. Representative immunofluorescence and quantification of (E and F) CD68 and (G and H) CD206 in microglia at P13 ( n = 6 mice per genotype, Scale bars: 50 μm). (I and J) Representative immunofluorescence and quantification of CD68 in microglia from adult Aurkb fl/fl and Cx3cr1 Cre/+ Aurkb fl/fl littermates ( n = 5 mice per genotype, Scale bars: 50 μm). The representative immunofluorescence image of the Cx3cr1 Cre/+ Aurkb fl/fl group is shared in C. (K and L) Adult Aurkb fl/fl and Cx3cr1 Cre/+ Aurkb fl/fl littermates were i.p. injected with LPS (1 mg/kg) and sacrificed at 48 h post LPS administration ( n = 5 mice per genotype, Scale bars: 50 μm). Data are presented as the mean ± SD. Two-tailed unpaired t-tests in (B, D, J, and I); two-way ANOVA with Bonferroni multiple comparisons test in (F, H); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant compared with the Aurkb fl/fl group. See also and .

Article Snippet: The following primary antibodies were used: rabbit anti-Iba-1 antibody (1:500, Wako, Cat: 019–19741), mouse anti-Iba-1 antibody (1:400, Abcam, Cat: ab283319), rat anti-CD206 antibody (1:200, Bio-Rad, Cat: MCA2235), rat anti-BrdU antibody (1:400, Abcam, Cat: ab6326), mouse anti-phospho-Histone H3 (Ser10) antibody (1:200, Cell Signaling Technology, Cat: 9706S), mouse anti-APC (1:100, CC-1, Merck, Cat: OP80), rat anti-myelin basic protein (Mbp) monoclonal antibody (1:500, Abcam, Cat: ab7349), rabbit anti-degraded myelin basic protein (dMbp) antibody (1:2000, Millipore Sigma, Cat: AB5864), rabbit anti-Olig2 antibody (1:500, Proteintech, Cat: 13999-1-AP) and rat anti-CD68 antibody (1:500, Abcam, Cat: ab53444).

Techniques: Injection, Immunofluorescence, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: EV-dead induces TNBC lung metastasis and promotes TAM infiltration in vivo. A Diagram of the EV-dead and EV-alive separation procedures and their representative transmission electron microscopy (TEM) images. Scale bar: 100 nm. The sizes of the EVs were detected using a Flow NanoAnalyzer, and their protein markers were identified by Western blotting analysis. B NTA analysis was conducted to detect the relative secretion content of EV-dead and EV-alive; n = 3. C Representative images of zebrafish breast cancer xenotransplantation model assay. The effects of EV-alive (100 μg/ml) and EV-dead (50–100 μg/ml) on the metastasis of 4 T1 cells in the presence or absence of M0 co-injection were investigated; n = 6. D Schematic diagram of the animal assay and representative pictures of the in vivo imaging assay and tumors. Peritumoral injection with EV-dead (200 μg/20 g weight, q3d) promoted tumor growth of 4 T1-Luc xenografts, as evidenced by increases in both tumor volume and weight, but had no significant effect on the weight of the mice; n = 8. Scale bar: 1 cm. E Representative images of the lungs and the lung HE staining assay. Metastatic foci were identified by HE staining of the lung sections. Scale bar: 100 μm; n = 3. F Infiltration levels of CD45 + /F4/80 + /CD206 + TAMs in mammary tumors; n = 3. G Expression levels of ARG1 and iNOS in mammary tumor tissues; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, Transmission Assay, Electron Microscopy, Western Blot, Injection, In Vivo Imaging, Staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: CXCL1 EV-dead promotes TNBC growth and lung metastasis in vivo by activating TAM/PD-L1 signaling. A The successful generation of 4 T1/rCXCL1 Flag cells was validated by immunoblotting and immunofluorescence assays. Scale bar: 5 μm. B The difference in CXCL1 content between EV-dead and EV-dead rCXCL1-Flag was compared by ELISA. EV-dead rCXCL1-Flag was isolated from the supernatants of apoptotic 4 T1/rCXCL1 Flag cells induced by paclitaxel treatment; n = 3. C , D Peritumoral injection with EV-dead rCXCL1 (200 μg/20 g weight, q3d) significantly accelerated TNBC growth (C) and lung metastasis (D) compared to that of the EV-dead group (200 μg/20 g weight, q3d); Tumor volume: n = 6; Number of metastatic lesions, n = 3; K-M curves of lung metastasis time, n = 10. Scale bar: 1 cm. E Tumor tissue immunofluorescence experiment showed that flag-tagged CXCL1 (red) from EV-dead rCXCL1-Flag was predominantly phagocytosed by CD206 + macrophages (green) in the TME. Scale bar: 5 μm. F The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs (up) and CD45 + /F4/80 + /PD-L1 + TAMs (down) in the TME of mice following treatment with saline, EV-dead, or EV-dead rCXCL1-Flag ; n = 3. G QPCR assay was conducted to investigate the CTC quantity in the peripheral blood of mice following treatment with saline, EV-dead, or EV-dead rCXCL1-Flag ; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Isolation, Injection, Saline

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: CXCL1 knockdown in EV-dead or macrophage depletion inhibits EV-dead-induced TNBC growth and lung metastasis in vivo . A The successful generation of 4 T1/shCXCL1 cells was verified by western blotting assay. The difference in CXCL1 content between EV-dead and EV-dead shCXCL1 was compared by ELISA. EV-dead shCXCL1 was isolated from the supernatants of apoptotic 4 T1/shCXCL1 cells induced by paclitaxel treatment; n = 3. B Representative images of the tumors ( n = 7) and the in vivo imaging assay ( n = 3), and mouse weight and tumor volume curves ( n = 7). Clodronate liposomes (CL) were used to deplete macrophages in the TME of the 4 T1-Luc xenograft model. Scale bar: 2 cm. C Representative images of the lungs ( n = 3) and lung HE assay ( n = 3) as well as the K-M curves of lung metastasis time ( n = 10). Scale bar: 100 μm. D , E The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs (D) and CD45 + /F4/80 + /PD-L1 + TAMs (E) in the TME of mice following treatment with EV-dead, EV-dead shCXCL1 , or the combination of EV-dead and CL; n = 3. F CD206 (green) and PD-L1 (red) expression levels in the TME. Arrows indicate PD-L1 expression in TAMs. Scale bar: 10 μm. G The quantity of CTCs in the peripheral blood of mice treated as indicated; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: Knockdown, In Vivo, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, In Vivo Imaging, Liposomes, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: TPCA-1 chemosensitizes TNBC to paclitaxel and inhibits CXCL1 EV-dead -induced TNBC growth and lung metastasis in vivo . A Representative images of tumors and the tumor volume curves ( n = 6). TPCA-1 (10 mg/kg/d) and paclitaxel (10 mg/kg/3d) were administered. B Representative images of the in vivo imaging assay and lung HE staining assay; n = 3. Scale bar: 100 μm. C , D The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs and CD45 + /F4/80 + /PD-L1 + TAMs in the TME of mice following treatment with TPCA-1, paclitaxel, or the combination of TPCA-1 and paclitaxel; n = 3. E The CTC quantity in the peripheral blood of mice when they were treated as indicated; n = 3. F Representative images of tumors and the tumor volume curves ( n = 6). EV-dead and EV-dead rCXCL1 (200 μg/20 g weight, q3d) were administered by peritumoral injection. G Representative images of the in vivo imaging assay and lung HE staining assay; n = 3. Scale bar: 100 μm. H , I The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs ( n = 3) and CD45 + /F4/80 + /PD-L1 + TAMs ( n = 6) in the TME of mice when they were treated as indicated. J The CTC quantity in the peripheral blood of mice when they were treated as indicated; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, In Vivo Imaging, Staining, Injection

Journal: STAR Protocols

Article Title: CyTOF protocol for immune monitoring of solid tumors from mouse models

doi: 10.1016/j.xpro.2022.101949

Figure Lengend Snippet:

Article Snippet: Anti-Mouse CD206-169Tm dilution 1:100 , Standard BioTools , 3169021B.

Techniques: Blocking Assay, Purification, Recombinant, Staining, Sterility, Antibody Labeling, Software, Transferring, Microscopy

Journal: Frontiers in Immunology

Article Title: Severe Fever With Thrombocytopenia Syndrome Virus-Induced Macrophage Differentiation Is Regulated by miR-146

doi: 10.3389/fimmu.2019.01095

Figure Lengend Snippet: SFTSV infection induced macrophage differentiation. (A) Real-time PCR analysis of M1 (TNF-α, IL-1β, IL-6, and CD86), and M2 markers (IL-10, CD163, CD206, and CCL22) of THP-1 cells infected for various time durations. (B) Flow-cytometry profiling of M1 (CD86 + ) and M2 (CD206 + ) macrophage subsets within a gated CD11b + population by FACS analysis. Percentage change of CD86 + cells (upper panel) and CD206 + cells (lower panel) is depicted. (C) Fluorescence analysis of M1 marker CD86 (green) and M2 marker CD206 (red) in SFTSV-infected THP-1 cells at various time points. Right graph: fluorescence intensity of CD86 (green) and CD206 (red) in the regions delineated by a white line through ImageJ software. In ImageJ software, pixel counts are used to determine the differences in fluorescence intensity. The value profiles represent fluorescence intensity of CD86 (green) and CD206 (red), which could reflect the expression levels of these two proteins. Bar = 50 μm. All data are shown as mean ± SEM of three independent experiments ( *** P < 0.001).

Article Snippet: Macrophages were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% TritonTM X-100 for 10 min, and blocked with 1% BSA for 1 h. The cells were then incubated with primary antibodies: rabbit anti-human CD86 (ab53004, Abcam, Cambridge, UK) at 1:500 dilution or mouse anti-human CD206 (170710, Bio-Rad, USA) at 1:200 dilution overnight at 4°C.

Techniques: Infection, Real-time Polymerase Chain Reaction, Flow Cytometry, Fluorescence, Marker, Software, Expressing

Journal: Frontiers in Immunology

Article Title: Severe Fever With Thrombocytopenia Syndrome Virus-Induced Macrophage Differentiation Is Regulated by miR-146

doi: 10.3389/fimmu.2019.01095

Figure Lengend Snippet: Macrophage differentiation affected SFTSV replication. (A) THP-1 cells are differentiated into M1 macrophages by 16 h incubation with 150 nM PMA and followed by incubating with IFN-γ and LPS, or into M2 with interleukin 4 and interleukin 13 for 24 h. Cells were then fixed and immunolabeled for CD86 or CD206 using specific antibodies. Nuclei were stained with DAPI (blue). Bar = 50 μm. (B) Western blot analysis of SFTSV nuclear protein (NP) in M1-like or M2-like cells after infection with SFTSV for 24 h at an MOI = 1. (C) Immunofluorescence staining of viral dsRNA (green) in M1-like or M2-like cells after 24 h infection. Nuclei were stained with DAPI (blue). Bar = 50 μm. (D,E) Quantitative real-time-PCR analysis of replicative copy number of SFTSV in the extracellular (D) or intracellular (F) compartments of the infected M1-like or M2-like cells. (F) Schematic presentation of the transwell co-culture with SFTSV-infected Hela cells in the top well and polarizing THP-1 cells in the bottom well. (G) Immunofluorescence staining of viral dsRNA (green) in Hela cells. Nuclei were stained with DAPI (blue). Bar = 50 μm. All data are presented as the mean ± SEM of three independent experiments. (B,C) ( ** P < 0.01, *** P < 0.001).

Article Snippet: Macrophages were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% TritonTM X-100 for 10 min, and blocked with 1% BSA for 1 h. The cells were then incubated with primary antibodies: rabbit anti-human CD86 (ab53004, Abcam, Cambridge, UK) at 1:500 dilution or mouse anti-human CD206 (170710, Bio-Rad, USA) at 1:200 dilution overnight at 4°C.

Techniques: Incubation, Immunolabeling, Staining, Western Blot, Infection, Immunofluorescence, Real-time Polymerase Chain Reaction, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Severe Fever With Thrombocytopenia Syndrome Virus-Induced Macrophage Differentiation Is Regulated by miR-146

doi: 10.3389/fimmu.2019.01095

Figure Lengend Snippet: miR-146 modulated SFTSV induced macrophage differentiation. (A) Schematic presentation of SFTSV infected THP-1 cell sorting. THP-1 was infected with SFTSV for 36 h at an MOI of 1, adhered cells re-suspended, washed once in RPMI and surface stained and sorted following standard Biolegend intracellular flow cytometry staining protocol. M1 macrophages are CD11b + CD86 + CD206 − cells, whereas M2 macrophages are CD11b + CD86 − CD206 + cells. (B) Representative heatmap of M1 and M2 cells through deep miRNA sequencing. (C,D) Real-time PCR analysis of miR-146a (C) and b (D) expression level in THP-1 cells at various time points. (E,F) Real-time PCR analysis of miR-146a/b expression in THP-1 cells after being transfected with miR-146a/b inhibitors or negative control (miR-inhibitors-n.c) for various durations. (G,H) Real-time-PCR analysis of CD86 or CD206 expression on THP-1 cells infected with SFTSV before transfection of miR-146a or b at various time points. (I,J) Expression levels of miR-146a (I) and 146b (J) were measured by real-time PCR in SFTSV infected patient sera. All the data are shown as mean ± SEM of three independent experiments ( * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Macrophages were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% TritonTM X-100 for 10 min, and blocked with 1% BSA for 1 h. The cells were then incubated with primary antibodies: rabbit anti-human CD86 (ab53004, Abcam, Cambridge, UK) at 1:500 dilution or mouse anti-human CD206 (170710, Bio-Rad, USA) at 1:200 dilution overnight at 4°C.

Techniques: Infection, FACS, Staining, Flow Cytometry, Sequencing, Real-time Polymerase Chain Reaction, Expressing, Transfection, Negative Control

Journal: Frontiers in Immunology

Article Title: Severe Fever With Thrombocytopenia Syndrome Virus-Induced Macrophage Differentiation Is Regulated by miR-146

doi: 10.3389/fimmu.2019.01095

Figure Lengend Snippet: Viral non-structural protein (NSs) contribute to miR-146b expression resulted in inducing M2 macrophage differentiation. (A) Western blot analysis of SFTSV non-structural protein (NSs) and miR-146a/b expression (B) in THP-1 cells that were transfected control pCMV3 plasmid, NSs over-expression plasmid (pCMV3-NSs) for 48 h, respectively. SFTSV-infected THP-1 cells as a positive control. (B,C) THP-1 cells were transfected pCMV3 plasmid or pCMV3-NSs for 48 h. The expression of miR-146a/b and endogenous STAT1 detected by real-time PCR and western blot, respectively. (D) Immunofluorescence staining analyzed for CD86 and CD206 expression after transfected with pCMV3 or pCMV3-NSs for 48 h. Images were acquired using a confocal microscope. Right graph: fluorescence intensity of CD86 (green) and CD206 (red) in the regions delineated by a white line through ImageJ software.

Article Snippet: Macrophages were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% TritonTM X-100 for 10 min, and blocked with 1% BSA for 1 h. The cells were then incubated with primary antibodies: rabbit anti-human CD86 (ab53004, Abcam, Cambridge, UK) at 1:500 dilution or mouse anti-human CD206 (170710, Bio-Rad, USA) at 1:200 dilution overnight at 4°C.

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Over Expression, Infection, Positive Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Microscopy, Fluorescence, Software

Journal: Frontiers in Immunology

Article Title: Severe Fever With Thrombocytopenia Syndrome Virus-Induced Macrophage Differentiation Is Regulated by miR-146

doi: 10.3389/fimmu.2019.01095

Figure Lengend Snippet: SFTSV induced macrophage differentiation in vivo . (A) . Flow-cytometry profiling of mouse splenic macrophages subsets after SFTSV infection. Macrophages were gated CD11b + and F4/80 + , M1 macrophages were gated CD11c + , and M2 macrophages were gated CD206 + . (B,C) The expression level of IL-10 (B) and IL-4 (C) were determined by ELISA in mouse serum that infected with SFTSV. (D,E) The expression of miR-146a (D) and miR-146b (E) within sorted mouse splenic macrophages in each group ( n = 3). Data in all panels are shown as mean ± SEM ( * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Macrophages were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% TritonTM X-100 for 10 min, and blocked with 1% BSA for 1 h. The cells were then incubated with primary antibodies: rabbit anti-human CD86 (ab53004, Abcam, Cambridge, UK) at 1:500 dilution or mouse anti-human CD206 (170710, Bio-Rad, USA) at 1:200 dilution overnight at 4°C.

Techniques: In Vivo, Flow Cytometry, Infection, Expressing, Enzyme-linked Immunosorbent Assay