Journal: Nature cell biology

Article Title: Immuno-subtyping of breast cancer reveals distinct myeloid cell profiles and immunotherapy resistance mechanisms.

doi: 10.1038/s41556-019-0373-7

Figure Lengend Snippet: Fig. 6 | TINs in NES tumours express multiple immunosuppressive pathways, and negatively regulate Ly6C+ monocyte recruitment. a, Heatmap showing unsupervised hierarchical clustering of TINs purified from four tumour models (n = 3 biologically independent samples for each model) using gene characteristic of normal neutrophils or TANs and/or MDSCs as published previously59. b, Heatmap showing unsupervised clustering of TINs of four tumour models (n = 3 biologically independent samples for each model) using GSVA of the 50 hallmark pathways from MSigDB. Pathways related to immunosuppressive activities are shown. The heatmap with all pathways annotated is shown in Supplementary Fig. 6a. c, In vitro immunosuppression assay by co-culturing bone marrow neutrophils from indicated NES-tumour-bearing animals and splenic T cells. Proliferation of T cells was determined based on CFSE intensity as measured by FACS. A left-shift of CFSE intensity histogram indicates dilution of signals by proliferation. Data are shown as mean ± s.d. of three biological replicates (neutrophils from three different mice). P values were determined by two-sided t-tests. d, Quantification of monocytes, TIN and TIM in NES tumours (PyMT-N and 2208L) following treatment of anti-CXCR2 and anti-Ly6G. Numbers in parentheses show the specific n values of biologically independent mice per group denoted by different colours. Data are shown as mean ± s.d. P values were computed by two-sided Student’s t-tests. e, Example FACS plots showing the alteration of TINs (CD11b+Ly6G+Ly6Cmed-low), monocytes (CD11b+Ly6G−Ly6C+), TIMs (CD11b+Ly6G−Ly6C−F4/80+) in hosts bearing PyMT-N tumours (NES) treated with anti-Ly6G and anti-CXCR2, or IgG control. The results are representative of at least five biologically independent animals.

Article Snippet: The following antibodies were used for FACS sorting as well as immune profiling: Myeloid cell phenotyping panel 1: CD45-violetFluor450 (clone 30-F11, Tonbo), CD11b-APC-Cy7 (clone M1/70, Tonbo), Ly6G-PerCPcy5.5 (clone 1A8, Tonbo), Ly6C-PE-CF594 (clone AL-21, BD Biosciences), F4/80-BV605 (clone BM8, Biolegend), I-A/I-E-BV510 (clone M5/114.15.2, Biolegend), CD11c-AlexaFluor700 (clone N418, Biolegend), CD64-APC (clone X54-5/7.1, Biolegend), CD103-PE-Cy7 (clone 2E7, Biolegend), PDL1-BV711 (clone MIH5, BD Biosciences) and DAPI (NucBlue Fixed Cell ReadyProbes Reagent).

Techniques: Purification, In Vitro, Immunosuppression Assay, Control

Journal: bioRxiv

Article Title: Hemozoin produced by mammals confers heme tolerance

doi: 10.1101/629725

Figure Lengend Snippet: Gating strategy of Ter-119 + cells in the (A) bone marrow and (D) spleen. Quantifications of total Ter-119 + cells in the (B) bone marrow and (E) spleen. The %single cells* on the y-axis denote single cells that are negative for CD4/8/41, B220 and Gr-1. C) Quantification of subpopulations of Ter-119 + cells represented as a percentage of total Ter-119 + cells in the bone marrow (n=7-12). F) Quantification of populations II and III of Ter-119 + cells represented as a percentage of total Ter-119 + cells in the spleen. Gating strategy (G, I) and quantification (H, J) of (G, H) splenic F4/80 hi Treml4 + red pulp macrophages (RPMs) and (I, J) F4/80 hi and F4/80 lo -CD11b hi splenic monocytes. At least 100,000 single cells were analyzed per sample. Each dot represents one mouse. ** p <0.05; ** p <0.01.

Article Snippet: For non-erythroid cell analysis, splenic cells were treated with RBC lysis buffer and stained with the following antibodies: phycoerythrin (PE)-conjugated Treml4 (Biolegend, San Diego, CA, cat. number 143304), APC-conjugated F4/80 (eBioscience, cat. number 17-4801-80), and PE-Cyanine7 (PE-Cy7)-conjugated CD11b (eBioscience, cat. number 25-0112-81).

Techniques:

Journal: bioRxiv

Article Title: Hemozoin produced by mammals confers heme tolerance

doi: 10.1101/629725

Figure Lengend Snippet: A) Kaplan-Meier survival curve of HRG1 +/+ and HRG1 -/- mice placed on a low-iron (2ppm) diet (n=15-17, both males and females). B-C) Hematocrits of HRG1 +/+ and HRG1 -/- mice placed on a standard or low-iron (2ppm) diet. Mice were placed on respective diets supplemented with deionized water starting at 21 days of age (week 0) (n=9-15 for 5-week data set; n=7-11 for 20-week data set). Quantification of tissue iron (D) and heme (E) by ICP-MS and UPLC respectively, in tissues of mice fed a low-iron (2ppm) diet (n=6-17). F) %Splenomegaly of HRG1 +/+ and HRG1 -/- mice calculated by the percentage of increase in average wet weight of spleens between mice on low-iron versus standard iron diets (n=9-15); G) Ratio of 2ppm splenic Ter-119 + population II+III cells to that of standard diet mice (n=8-12); H) Quantification of total Ter-119 + cells in the spleen. The %single cells* on the y-axis denote single cells that are negative for CD4/8/41, B220 and Gr-1. Each point represents one mouse. I) Quantification of subpopulations of Ter-119 + cells represented as a percentage of total Ter-119 + cells in the bone marrow (n=7-8); J) Quantifications of splenic RPMs in mice on a standard or low-iron (2ppm) diet, represented as a percentage of single cells analyzed (n=9-14). K) Quantification of the ratio of F4/80 hi to F4/80 lo CD11b hi splenic monocytes from mice on a standard or low-iron (2ppm) diet (n= 9-15). At least 100,000 single cells were analyzed per sample. L) Gene expression heat map of 90 iron metabolism genes in spleens from mice on standard or low-iron (2ppm) diet. Pearson correlation was used for comparison; average linkage (n=9 per group, per genotype). * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001.

Article Snippet: For non-erythroid cell analysis, splenic cells were treated with RBC lysis buffer and stained with the following antibodies: phycoerythrin (PE)-conjugated Treml4 (Biolegend, San Diego, CA, cat. number 143304), APC-conjugated F4/80 (eBioscience, cat. number 17-4801-80), and PE-Cyanine7 (PE-Cy7)-conjugated CD11b (eBioscience, cat. number 25-0112-81).

Techniques: Expressing

Journal: European Journal of Medical Research

Article Title: Dok-3 deficient mice display different immune clustering and Tim-3 expression

doi: 10.1186/s40001-019-0384-7

Figure Lengend Snippet: Macrophages and MDSCs proportion and the Tim-3 expression on these cells in two group mice. Spleen immune cells were isolated from 129 mice ( n = 6) and Dok-3 −/− mice ( n = 6). a Flow cytometry (FCM) analysis of Tim-3 expression on macrophages marked by CD3 − CD11b + (left), and the statistical graph was shown (right), macrophages (129, 12.33 ± 2.131; Dok-3 −/− , 31.67 ± 3.336) and Tim-3 expression (129, 28.61 ± 4.120; Dok-3 −/− , 16.18 ± 1.874). b Flow cytometry (FCM) analysis of Tim-3 expression on MDSCs marked by CD3 − CD11b + (left), and the statistical graph was shown (right), MDSCs (129, 9.72 ± 1.886; Dok-3 −/− , 23.73 ± 3.150) and Tim-3 expression (129, 19.58 ± 1.661; Dok-3 −/− , 12.58 ± 1.315). Both the macrophages and MDSCs’ gated strategy are shown in Additional file : Figure S1. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The cells were stained with anti-mouse Tim-3-PE (ebioscience), anti-mouse CD3-APC (ebioscience) or anti-mouse CD3-FITC (ebioscience) which was used in DC cells marking, anti-mouse CD4-FITC (ebioscience), anti-mouse CD8-FITC (ebioscience), anti-mouse CD11b-Pe-cy-7 (ebioscience), anti-mouse NK1.1-PerCP-5.5 (ebioscience), anti-mouse CD11c-APC (ebioscience), anti-mouse Gr-1-FICT (ebioscience) for 30 min. At least 10,000 cells were analyzed by a FACSAriaII.

Techniques: Expressing, Isolation, Flow Cytometry

Journal: International Journal of Environmental Research and Public Health

Article Title: The Effects of Indoor Pollutants Exposure on Allergy and Lung Inflammation: An Activation State of Neutrophils and Eosinophils in Sputum

doi: 10.3390/ijerph17155413

Figure Lengend Snippet: Gating strategy to identify activated sputum granulocytes. ( A ) A forward-/side-scatter (FSC/SSC) gate to identify the granulocytes. ( B ) The activated granulocytes were based on CD11b+. ( C ) CD41a was gated with SSC to confirm that the activated granulocytes were neutrophils and eosinophils. ( D ) Subsequently, the eosinophils (blue) and neutrophils (green) were separated based on the expression of CD16− and CD16+ on SSC, respectively. The activation markers of CD11b, CD35, CD63, and CD66b for eosinophils ( E , F ) and neutrophils ( G , H ) were based on the negative gates of the isotype control for all antibodies.

Article Snippet: The cells were immunostained with antibodies for neutrophil and eosinophil surface markers of PE-Cy 7 Mouse Anti-Human CD11b/Mac-1, BB515 Mouse Anti-Human CD35, PE Mouse Anti-Human CD63, APC-H7 Mouse Anti-Human CD16, PerCP-Cy 5.5 CD41a, and Alexa Fluor 647 Mouse Anti-Human CD66b purchased from BD Biosciences, US.

Techniques: Expressing, Activation Assay

Journal: International Journal of Environmental Research and Public Health

Article Title: The Effects of Indoor Pollutants Exposure on Allergy and Lung Inflammation: An Activation State of Neutrophils and Eosinophils in Sputum

doi: 10.3390/ijerph17155413

Figure Lengend Snippet: Expression profile of sputum granulocytes compared between the doctor-diagnosed asthmatic and healthy school children. Expression of degranulation and activation markers on eosinophil ( A , B ) and neutrophil ( C , D ). The expression of CD11b as the degranulation and classical activation marker is displayed twice in this graph. * p < 0.05, ** p < 0.001.

Article Snippet: The cells were immunostained with antibodies for neutrophil and eosinophil surface markers of PE-Cy 7 Mouse Anti-Human CD11b/Mac-1, BB515 Mouse Anti-Human CD35, PE Mouse Anti-Human CD63, APC-H7 Mouse Anti-Human CD16, PerCP-Cy 5.5 CD41a, and Alexa Fluor 647 Mouse Anti-Human CD66b purchased from BD Biosciences, US.

Techniques: Expressing, Activation Assay, Marker

Journal: International Journal of Environmental Research and Public Health

Article Title: The Effects of Indoor Pollutants Exposure on Allergy and Lung Inflammation: An Activation State of Neutrophils and Eosinophils in Sputum

doi: 10.3390/ijerph17155413

Figure Lengend Snippet: Factor loadings using PCA for eosinophils and eosinophils. The moderate (0.5–0.75) and strong (> 0.75) factor loadings are highlighted in bold.

Article Snippet: The cells were immunostained with antibodies for neutrophil and eosinophil surface markers of PE-Cy 7 Mouse Anti-Human CD11b/Mac-1, BB515 Mouse Anti-Human CD35, PE Mouse Anti-Human CD63, APC-H7 Mouse Anti-Human CD16, PerCP-Cy 5.5 CD41a, and Alexa Fluor 647 Mouse Anti-Human CD66b purchased from BD Biosciences, US.

Techniques:

Journal: International Journal of Environmental Research and Public Health

Article Title: The Effects of Indoor Pollutants Exposure on Allergy and Lung Inflammation: An Activation State of Neutrophils and Eosinophils in Sputum

doi: 10.3390/ijerph17155413

Figure Lengend Snippet: Summary of the binary logistic regression models based on the clusters generated in the AHC analysis.

Article Snippet: The cells were immunostained with antibodies for neutrophil and eosinophil surface markers of PE-Cy 7 Mouse Anti-Human CD11b/Mac-1, BB515 Mouse Anti-Human CD35, PE Mouse Anti-Human CD63, APC-H7 Mouse Anti-Human CD16, PerCP-Cy 5.5 CD41a, and Alexa Fluor 647 Mouse Anti-Human CD66b purchased from BD Biosciences, US.

Techniques: Generated