Journal: Cell Reports Medicine

Article Title: Endocrine-targeting therapies shift the breast microbiome to reduce estrogen receptor-α breast cancer risk

doi: 10.1016/j.xcrm.2024.101880

Figure Lengend Snippet:

Article Snippet: Pierce™ Bovine Serum Albumin Standard Pre-Diluted Set , ThermoFisher Scientific , Cat #: 23208.

Techniques: Recombinant, Virus, Sequencing, Software, Migration, Real-time Polymerase Chain Reaction, Imaging, SYBR Green Assay, Reverse Transcription, Control, Western Blot, Blocking Assay, Immunohistochemistry, Stripping, Staining

Journal: Cell Stem Cell

Article Title: Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response

doi: 10.1016/j.stem.2019.02.019

Figure Lengend Snippet:

Article Snippet: RNase-Free DNase Set , QIAGEN , Cat#79254.

Techniques: Blocking Assay, Purification, Recombinant, CRISPR, Electroporation, Negative Control, Staining, SYBR Green Assay, Multiplex Assay, Flow Cytometry, cDNA Synthesis, Software, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Frontiers in immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: FIGURE 1 | Elevated lipocalin 2 (Lcn2) during E. coli O157:H7 infection. (A) Real-time PCR analysis of the levels of Lcn2 mRNA expression in indicated tissues of mice. (B) The mRNA levels of Lcn2 expression in indicated tissues of control and E. coli O157:H7-infected mice. (C) Serum levels of Lcn2 protein concentration in E. coli O157:H7-infected mice after challenge at different time points. (D–G) The mRNA expression levels of Lcn2 in indicated tissues from E. coli O157:H7-infected mice after challenge at different time points. (H) Protein levels of Lcn2 in mice detected on immunohistochemistry sections of liver and jejunum at 8 and 32 h after challenge with E. coli O157:H7. Original magnification was 200×. Values are average means of triplicate experiments. Error bars depict SEM (n = 4 per time point). Results are expressed as means ± SEM. *P < 0.05, and **P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer’s instructions.

Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing, Control, Protein Concentration, Immunohistochemistry

Journal: Frontiers in immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: FIGURE 2 | The bacteriostatic characteristics of lipocalin 2 (Lcn2). (A,B) Bacterial loads in blood (CFU/ml) and livers (CFU/mg) of E. coli O157:H7-infected mice 32 hpi. (C,D) Serum levels of Lcn2 protein measured and growth of E. coli O157:H7 in RPMI with 20% acute-phase serum from wild-type (WT) or Lcn2−/−mice. Values are average means of triplicate experiments with two mice per genotype per experiment. Error bars depict SEM (n = 6 per group). Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. *P < 0.05.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer’s instructions.

Techniques: Infection

Journal: Frontiers in immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: FIGURE 3 | Granulocyte abnormalities in Lcn2−/−mice. (A) Hematological parameters of peripheral blood from wild-type (WT) and Lcn2−/−mice. The data are presented as mean × 103 cells/µl. Error bars depict SEM (n = 6 per group). (B) Flow cytometry analysis of neutrophils in the peripheral blood after intragastric administration with 2 × 108 CFU of E. coli O157:H7. Cells were stained with indicated clones of Gr-1 Ab, and positive cells were determined by flow cytometry. Values are average means of triplicate experiments with two mice per genotype per experiment. Error bars depict SEM. (C) Wright-Giemsa staining of peripheral blood smears from Lcn2−/−mice identified atypical hyposegmented neutrophils in the peripheral blood. Original magnification ×63. In contrast, WT mice displayed normal neutrophil maturation. (D) Enumeration of the number of band neutrophils in the peripheral blood of Lcn2−/−mice. The data are presented as mean band cell numbers per 100 leukocytes. Error bars depict SEM. Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. *P < 0.05, and **P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Staining, Clone Assay, Cytometry

Journal: Frontiers in immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: FIGURE 4 | Reduced migration of lipocalin 2-deficient (Lcn2−/−) neutrophils. (A,B) Flow cytometry analysis of peripheral blood and peritoneal exudates of heat-killed E. coli O157:H7-challenged mice following staining with a Gr-1 PE Ab. (C,D) ELISA analysis of TNF-α in the serum and peritoneal exudates of heat-killed E. coli O157:H7-challenged mice. (E,F) Quantitative determination of chemokines MCP-1 and MIP-2 mRNA expression in the liver of heat-killed E. coli O157:H7-challenged mice. Values are average means of triplicate experiments with two mice per genotype per experiment. Error bars depict SEM. Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. *P < 0.05 and **P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer’s instructions.

Techniques: Migration, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: FIGURE 5 | Decreased expression of inflammatory cytokines produced by lipocalin 2-deficient (Lcn2−/−) macrophages. (A) Real-time PCR analysis of Lcn2 mRNA expression levels in the E. coli O157:H7-infected primary bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Lcn2−/−mice. (B–D) ELISA analysis of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels in the culture medium of the E. coli O157:H7-infected BMDMs from WT and Lcn2−/−mice. (E–L) Real-time PCR analysis of cytokine mRNA expression levels in the E. coli O157:H7-infected pBMDMs from WT and Lcn2−/−mice. (M) The infected BMDMs of WT and Lcn2−/−mice were subjected to staining with rabbit monoclonal antibody iNOS, rat monoclonal antibody F4/80, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 647 goat anti-rat IgG in blocking buffer (1:200) and observed by fluorescence microscopy. Values are average means of triplicate experiments with two mice used for the isolation of BMDMs per genotype per experiment. Error bars depict SEM. Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. *P < 0.05 and **P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer’s instructions.

Techniques: Expressing, Produced, Real-time Polymerase Chain Reaction, Infection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining, Blocking Assay, Microscopy, Isolation

Journal: Frontiers in immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: FIGURE 6 | Lipocalin 2 (Lcn2) can promote migration and phagocytosis of macrophages. (A,B) Scratch wound healing assay of mouse RAW264.7 macrophages and quantification of the fold change of average migrated distance of cells was measured with microscope (n = 3, mean ± SEM, scale bars, 100 µm). (C,D) ELISA analysis of monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 levels in the culture medium of Lcn2-treated macrophages. (E) Flow cytometry analysis of mouse RAW264.7 macrophages incubated with FITC-dextran. Values are average means of triplicate experiments with two repetitions per treatment per experiment. Error bars depict SEM. *P < 0.05 and **P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer’s instructions.

Techniques: Migration, Wound Healing Assay, Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells

doi: 10.3390/ijms231810300

Figure Lengend Snippet: Identification of HRG as a binding protein to S100A8/A9. ( A ) Recombinant proteins (GST, GST-S100A8, GST-S100A9) were prepared from the E. coli expression system. Ten micrograms of each individual single protein or a preincubated mixture of GST-S100A8 and GST-S100A9 (GST-S100A8/A9) was incubated with human plasma (1 mL) collected from a healthy volunteer donor. After incubation for 30 min at room temperature under gentle shaking, the GST proteins were all pulled down by the addition of glutathione-conjugated Sepharose beads. After the beads were washed extensively, the bound proteins were eluted with glutathione and subjected to SDS-PAGE for analysis. The Coomassie brilliant blue (CBB)-stained gel displayed three clear bands (BP1, 2, and 3) that co-shed with GST-S100A9 and GST-S100A8/A9. ( B ) Interaction of S100A8/A9 with HRG was confirmed by ELISA. A 96-well plate coated with the highly purified human recombinant HRG protein (100 µg/mL) was incubated with the indicated concentrations of biotinylated human recombinant S100A8/A9 protein after blocking with a chemical-based reagent, Blockmaster DB1130, that is very good for quenching the nonspecific binding of S100A8/A9. The binding of S100A8/A9 to HRG was detected by treatment with HRP-conjugated streptavidin and the chemical reaction between the HRP and the substrate used. The background values of S100A8/A9 binding from the wells without HRG coating were deducted. Data are expressed as optical density (O.D.) means ± SD. *** p < 0.001 by Student’s t -test. ( C ) Schematic drawing of the method used to detect the interaction between S100 family proteins and HRG on the HEKT293 cell surface (left). To effectively increase the probability of interaction between S100 proteins and HRG in the extracellular space, all proteins were modified to express in a membrane-anchored form, so that their density was much greater on the narrow cell surface than in the vast extracellular space. HEK293T cells were co-transfected with myc-tagged modified HRG and HA-tagged S100 family-expressing vectors. After the immunoprecipitation of the expressed cell-surface HRG with myc antibody-conjugated beads, the interacting cell surface S100 proteins were detected by the HA antibody (right).

Article Snippet: Human S100A8/A9 and HRG ELISA were performed using a sandwich procedure under conventional conditions using a mouse anti-human S100A8/A9 antibody (clones #45 and #260) developed by our group, as reported previously, and a commercially available HRG-ELISA kit (human HPRG/HRG ELISA pair set; Sino Biological, Beijing, China).

Techniques: Binding Assay, Recombinant, Expressing, Incubation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Modification, Transfection, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells

doi: 10.3390/ijms231810300

Figure Lengend Snippet: Profiling of the expression pattern of mouse S100 family genes in the premetastatic stage in the lungs and brain of C57BL/6J mice burdened with melanoma (B16-BL6 cells). ( A ) Lungs and brain were resected from C57BL/6J mice burdened with melanoma (B16-BL6 cells) on day 7 after inoculation of melanoma to the subcutaneous area of the ear. ( B ) Total RNAs prepared from the indicated organs (lungs and brain) were analyzed for the expression of S100 family genes by quantitative real-time PCR. Tbp mRNA was used as a control for the analysis. The data were expressed as an expression ratio in comparison with the respective values from the healthy lungs and brain of nontumorous control mice, which were set as 1. ( C ) S100A8/A9 and HRG levels in the plasma collected from brain-metastatic lung cancer patients. The backgrounds of patients are described in Materials and Methods. S100A8/A9 and HRG levels were measured with ELISA. Data in panels ( B , C ) are means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test.

Article Snippet: Human S100A8/A9 and HRG ELISA were performed using a sandwich procedure under conventional conditions using a mouse anti-human S100A8/A9 antibody (clones #45 and #260) developed by our group, as reported previously, and a commercially available HRG-ELISA kit (human HPRG/HRG ELISA pair set; Sino Biological, Beijing, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: STAR Protocols

Article Title: Protocol for influenza A virus infection of mice and viral load determination

doi: 10.1016/j.xpro.2022.101151

Figure Lengend Snippet:

Article Snippet: Influenza A H1N1 (A/Puerto Rico/8/1934) Hemagglutinin/HA ELISA Pair Set , Sino Biological , Cat# SEK11684.

Techniques: Recombinant, Injection, Purification, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software, Hybridization, Blocking Assay, Spectrophotometry, Real-time Polymerase Chain Reaction, Microplate Reader Absorbance Measurement, Inverted Microscopy, Flow Cytometry, Plasmid Preparation, Laser-Scanning Microscopy

Journal: Arthritis Research & Therapy

Article Title: The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

doi: 10.1186/ar1946

Figure Lengend Snippet: Summary of primers used

Article Snippet: To detect mouse IL-1F8, a similar ELISA was set up using a polyclonal anti-mouse IL-1F8 antibody, a biotin-conjugated polyclonal anti-mouse IL-1F8 antibody and recombinant mouse IL-1F8 (R&D Systems).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

doi: 10.1186/ar1946

Figure Lengend Snippet: Production of IL-6, IL-8 and nitric oxide by hSFs and hACs: effects of IL-1F8 and IL-1β. Shown is an analysis of the effects of IL-1F8 and IL-1β on production of (a) IL-6 and (c) IL-8 by hSFs, and of (b) IL-6, (d) IL-8 and (e) nitric oxide by hACs. Cells were treated with the indicated cytokine concentrations for 48 hours, as detailed under Materials and method. * P < 0.05 versus control; # P < 0.05 versus 0.1 ng/ml IL-1β; & P < 0.05 versus 500 ng/ml IL-1F8, determined using analysis of variance. IL, interleukin; hAC, human articular chondrocyte; hSF, human synovial fibroblast.

Article Snippet: To detect mouse IL-1F8, a similar ELISA was set up using a polyclonal anti-mouse IL-1F8 antibody, a biotin-conjugated polyclonal anti-mouse IL-1F8 antibody and recombinant mouse IL-1F8 (R&D Systems).

Techniques: Control

Journal: Arthritis Research & Therapy

Article Title: The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

doi: 10.1186/ar1946

Figure Lengend Snippet: Production of IL-6 by hACs: effects of IL-1β, IL-1F8, heat-inactivated IL-1F8 and anti-IL-1Rrp2 antibodies. (a) Analysis of the effects of IL-1β (1 ng/ml), IL-1F8 and heat-inactivated IL-1F8 (5 mg/ml) on IL-6 production by hACs after 48 hours treatment, as detailed under Materials and method. (b) Analysis of the effects of anti-IL-1Rrp2 antibodies on IL-6 production by hACs. Cells were stimulated or not (control) for 48 hours with IL-1F8 (5 mg/ml) or IL-1b (1 ng/ml), as indicated, in the presence (black columns) or absence (white columns) of blocking anti-IL-1Rrp2 antibodies (10 mg/ml). P < 0.05 versus control; # P < 0.05 versus 5 μg/ml IL-1F8, as determined by analysis of variance. hAC, human articular chondrocyte; IL, interleukin; IL-1Rrp2, IL-1 receptor related protein 2.

Article Snippet: To detect mouse IL-1F8, a similar ELISA was set up using a polyclonal anti-mouse IL-1F8 antibody, a biotin-conjugated polyclonal anti-mouse IL-1F8 antibody and recombinant mouse IL-1F8 (R&D Systems).

Techniques: Control, Blocking Assay

Journal: Arthritis Research & Therapy

Article Title: The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

doi: 10.1186/ar1946

Figure Lengend Snippet: Kinetics of IL-1β and IL-1F8 mRNA production by HSFs and hCAs in response to IL-1 and/or TNF-α. (a) Analysis of IL-1F8 mRNA levels in hSFs treated or not treated for 8 hours with IL-1β (1 ng/ml) and/or TNF-α (10 ng/ml), as detailed under Materials and method. A representative agarose gel electrophoresis of PCR products is shown. (b) Real-time PCR analysis of IL-1F8 mRNA levels in hSFs stimulated (black columns) or not stimulated (white columns) for 8 hours with IL-1β (1 ng/ml) alone, IL-1β (1 ng/ml) plus TNF-α (10 ng/ml), or IL-1α (1 ng/ml) alone, as indicated. The amount of 28S rRNA was monitored as an internal control. The expression of IL-1F8 mRNA was corrected for 28S rRNA levels and the IL-1F8/28S ratios were normalized to the maximal value observed in each experiment, which was set to 100%. The results shown represent the mean ± standard error of data obtained with samples from three independent cultures. * P < 0.05 versus, as determined by analysis of variance. (c) Fold increase (after correction for 28S RNA levels) in IL-1β (dashed line) and IL-1F8 (solid line) mRNA levels after treatment of hSFs with 1 ng/ml IL-1β for the indicated times, as revealed by real-time PCR analysis. Basal IL-1F8/28S and IL-1β/28S levels were respectively 6.4 and 20 (arbitrary units). (d) Real-time PCR analysis of IL-1F8 and IL-1β mRNA levels in hACs stimulated (black columns) or not stimulated (white columns) with IL-1β (1 ng/ml) and TNF-α (10 ng/ml) for 8 hours. The amount of 28S rRNA was monitored as an internal control. The expression of IL-1F8 and IL-1β mRNA was corrected for 28S rRNA levels and the IL/28S ratios were normalized to the maximal value observed in each experiment, which was set to 100%. The results shown represent the mean ± standard error of data obtained with samples from six independent cultures. * P < 0.05 versus, as determined by analysis of variance. hAC, human articular chondrocyte; hSF, human synovial fibroblast; IL, interleukin; RT-PCR, reverse transcriptase polymerase chain reaction; TNF, tumour necrosis factor.

Article Snippet: To detect mouse IL-1F8, a similar ELISA was set up using a polyclonal anti-mouse IL-1F8 antibody, a biotin-conjugated polyclonal anti-mouse IL-1F8 antibody and recombinant mouse IL-1F8 (R&D Systems).

Techniques: Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

doi: 10.1186/ar1946

Figure Lengend Snippet: IL-1F8 protein levels in mouse and human joint samples and in mouse skin

Article Snippet: To detect mouse IL-1F8, a similar ELISA was set up using a polyclonal anti-mouse IL-1F8 antibody, a biotin-conjugated polyclonal anti-mouse IL-1F8 antibody and recombinant mouse IL-1F8 (R&D Systems).

Techniques: Control

Journal: Arthritis Research & Therapy

Article Title: The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

doi: 10.1186/ar1946

Figure Lengend Snippet: IL-1F8 protein levels in control individuals, patients with RA, OA and septic shock. Shown are serum IL-1F8 protein levels in healthy donors ( n = 16), patients with RA ( n = 28) or OA ( n = 16) patients, and patients with septic shock ( n = 12), as determined by ELISA. Individual values (grey dots) and mean (stippled lines) ± standard error (black lines) are shown. Differences between the groups were not significant. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; OA, osteoarthritis; RA, rheumatoid arthritis.

Article Snippet: To detect mouse IL-1F8, a similar ELISA was set up using a polyclonal anti-mouse IL-1F8 antibody, a biotin-conjugated polyclonal anti-mouse IL-1F8 antibody and recombinant mouse IL-1F8 (R&D Systems).

Techniques: Control, Enzyme-linked Immunosorbent Assay