Journal: Cell reports

Article Title: β-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia

doi: 10.1016/j.celrep.2020.02.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Thereafter, membranes were blocked with 3% fatty acid-free bovine serum albumin (BSA) (Millipore) in Tris-buffered saline supplemented with Tween-20 (TBST) (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 8.0) for 1 h at RT, followed by incubation with the primary antibodies mouse anti-NLRP3 (1:1000, AdipoGen), rat anti-caspase-1 (1:1000, clone 4B4, Genentech), rabbit anti-ASC (1:1000; clone AL177, AdipoGen), rabbit anti-GSDMD (1:1000, Abcam), goat anti-TREM2 (1:1000, GeneTex), mouse anti-Aβ (82E1) (1:1000, IBL America), rabbit-anti-IL-1β (1:500, GeneTex), rabbit anti-GAPDH (1:1000, Sigma-Aldrich) and rabbit anti-β-actin (1:1000, Cell Signaling Technology®), O/N at 4°C, respectively.

Techniques: Control, Recombinant, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Quantitation Assay, Gel Extraction, Purification, Isolation, Software, Transmission Assay, Microscopy, Spectrophotometry, Fluorescence, Imaging

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory monocytes drive influenza A virus-mediated lung injury in juvenile mice

doi: 10.4049/jimmunol.1701543

Figure Lengend Snippet: Antibodies used for Western blot

Article Snippet: Signals were detected following incubation with IRDye® Secondary Antibodies (LI-COR Biosciences, 1:10,000) for two hours at room temperature using the LI-COR Odyssey Fc Imaging System® ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody target Manufacturer Class Clone Secondary Antibody NLRP3 Adipogen monoclonal Cryo-2 Donkey anti-mouse Caspase-1 Santa Cruz monoclonal 14F468 Donkey anti-rabbit ASC (AL177) Adipogen polyclonal Donkey anti-rabbit IL-18 ( {"type":"entrez-protein","attrs":{"text":"P70380","term_id":"3219810","term_text":"P70380"}} P70380 ) Biovision polyclonal Donkey anti-rabbit Actin (C-11) Santa Cruz polyclonal Donkey anti-goat Open in a separate window Antibodies used for Western blot Submandibular bleeding Approximately 150 µl of blood was collected from the submandibular vein in an EDTA-coated tube (BD) for flow cytometry analysis or in a Microtainer Tube with Serum Separator (BD) for ELISA analysis.

Techniques: Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory monocytes drive influenza A virus-mediated lung injury in juvenile mice

doi: 10.4049/jimmunol.1701543

Figure Lengend Snippet: Juvenile and adult mice were infected with IAV (25 PFU WSN i.t.). A–B) TNF-α and IL-6 levels in BALF were measured by ELISA on days 0, 3, 5, and 7 p.i. (**)p<0.01, (****)p<0.0001, adult versus juvenile by two-way ANOVA with a correction provided by the Sidak multiple comparisons test. Lung homogenates from IAV-infected adult and juvenile mice were assessed by Western blot for C) NLRP3 D) ASC and E) pro-caspase-1. (*)=p<0.05, (**)p<0.01, PBS versus IAV or juvenile versus adult by two-way ANOVA with a correction provided by the Sidak multiple comparisons test. F–G) Mature Caspase-1 (mCaspase-1) and Mature IL-18 (mIL-18) were measured by ELISA on days 0, 3, 5, and 7 p.i. Lung homogenates from IAV-infected adult and juvenile mice were assessed by Western blot for H) mCaspase-1 and I) mIL-18. Data in A-I are from 2 independent experiments of n=5–9 mice per group and presented as mean +/− SD. (*)=p<0.05, (***)p<0.001, (****)p<0.0001, adult versus juvenile by two-way ANOVA with a correction provided by the Sidak multiple comparisons test.

Article Snippet: Signals were detected following incubation with IRDye® Secondary Antibodies (LI-COR Biosciences, 1:10,000) for two hours at room temperature using the LI-COR Odyssey Fc Imaging System® ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody target Manufacturer Class Clone Secondary Antibody NLRP3 Adipogen monoclonal Cryo-2 Donkey anti-mouse Caspase-1 Santa Cruz monoclonal 14F468 Donkey anti-rabbit ASC (AL177) Adipogen polyclonal Donkey anti-rabbit IL-18 ( {"type":"entrez-protein","attrs":{"text":"P70380","term_id":"3219810","term_text":"P70380"}} P70380 ) Biovision polyclonal Donkey anti-rabbit Actin (C-11) Santa Cruz polyclonal Donkey anti-goat Open in a separate window Antibodies used for Western blot Submandibular bleeding Approximately 150 µl of blood was collected from the submandibular vein in an EDTA-coated tube (BD) for flow cytometry analysis or in a Microtainer Tube with Serum Separator (BD) for ELISA analysis.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory monocytes drive influenza A virus-mediated lung injury in juvenile mice

doi: 10.4049/jimmunol.1701543

Figure Lengend Snippet: Juvenile and adult mice were infected with IAV (25 PFU WSN i.t.). CD45+ cells were isolated from lung homogenates 7 days p.i. and intrapulmonary immune cells were identified via flow cytometry. A) Total number of neutrophils (Ly6G+, CD11b+, CD24+), eosinophils (CD11b+, CD24+, Siglec F+, CD11c–, CD64–), recruited monocytes (CD11b+, Ly6C+, CD64+/−), and alveolar macrophages (CD64+, CD11c+, Siglec F+) per dry lung weight as determined by flow cytometry of lung homogenates. Data are from 3 independent experiments of n=4–8 mice per group and presented as mean +/− SD. (*) p<0.05 juvenile versus adult as assessed by multiple T-tests with a correction provided by the Holm-Sidak multiple comparisons test. B–E) Intracellular immunofluorescent antibody staining for caspase-1 and NLRP3 in Ly6C+CD64+/− cells analyzed by flow cytometry. Data represented by histogram with fluorescence minus one (FMO) control (B and D) or the median fluorescent intensity (MFI) (C and E). Data in B-E are from 2 independent experiments of n=3–4 mice per group and presented as mean +/− SD. (*) p<0.05, adult versus juvenile (A) or PBS versus IAV and adult versus juvenile (C and E) by two-way ANOVA with a correction provided by the Sidak multiple comparisons test. Neut = neutrophils, eos = eosinophils, MoDCs = recruited monocytes, AMs = alveolar macrophages, FMO = fluorescence minus one.

Article Snippet: Signals were detected following incubation with IRDye® Secondary Antibodies (LI-COR Biosciences, 1:10,000) for two hours at room temperature using the LI-COR Odyssey Fc Imaging System® ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody target Manufacturer Class Clone Secondary Antibody NLRP3 Adipogen monoclonal Cryo-2 Donkey anti-mouse Caspase-1 Santa Cruz monoclonal 14F468 Donkey anti-rabbit ASC (AL177) Adipogen polyclonal Donkey anti-rabbit IL-18 ( {"type":"entrez-protein","attrs":{"text":"P70380","term_id":"3219810","term_text":"P70380"}} P70380 ) Biovision polyclonal Donkey anti-rabbit Actin (C-11) Santa Cruz polyclonal Donkey anti-goat Open in a separate window Antibodies used for Western blot Submandibular bleeding Approximately 150 µl of blood was collected from the submandibular vein in an EDTA-coated tube (BD) for flow cytometry analysis or in a Microtainer Tube with Serum Separator (BD) for ELISA analysis.

Techniques: Infection, Isolation, Flow Cytometry, Staining, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory monocytes drive influenza A virus-mediated lung injury in juvenile mice

doi: 10.4049/jimmunol.1701543

Figure Lengend Snippet: Juvenile mice infected with IAV produce more IFN-αβ, secrete more MCP-1, recruit more inflammatory monocytes, and have increased activation of the NLRP3 inflammasome compared to adult mice. These differences contribute to increased IAV-induced lung injury in juvenile mice.

Article Snippet: Signals were detected following incubation with IRDye® Secondary Antibodies (LI-COR Biosciences, 1:10,000) for two hours at room temperature using the LI-COR Odyssey Fc Imaging System® ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody target Manufacturer Class Clone Secondary Antibody NLRP3 Adipogen monoclonal Cryo-2 Donkey anti-mouse Caspase-1 Santa Cruz monoclonal 14F468 Donkey anti-rabbit ASC (AL177) Adipogen polyclonal Donkey anti-rabbit IL-18 ( {"type":"entrez-protein","attrs":{"text":"P70380","term_id":"3219810","term_text":"P70380"}} P70380 ) Biovision polyclonal Donkey anti-rabbit Actin (C-11) Santa Cruz polyclonal Donkey anti-goat Open in a separate window Antibodies used for Western blot Submandibular bleeding Approximately 150 µl of blood was collected from the submandibular vein in an EDTA-coated tube (BD) for flow cytometry analysis or in a Microtainer Tube with Serum Separator (BD) for ELISA analysis.

Techniques: Infection, Activation Assay

Journal: The EMBO Journal

Article Title: Tissue‐resident macrophages actively suppress IL‐1beta release via a reactive prostanoid/IL‐10 pathway

doi: 10.15252/embj.2019103454

Figure Lengend Snippet: A Normalized expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell‐sorted Gata6‐WT and Gata6‐KO mye pMФ. Data are shown as mean ± SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamini–Hochberg procedure. n.d = non‐detectable. B Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6‐WT and Gata6‐KO mye pMФ. n = 4–7 individual mice per group. C, D Il1b and Tnf mRNA relative expression (C) and IL‐1β Western blot protein analysis (D) of Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS for 3 and 6 h respectively. Data shown are representative of at least three independent experiments. Western blot was performed on whole cell lysates. E Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro‐IL‐1β + Gata6‐WT and Gata6‐KO mye pMФ flow cytometry analysis 3 h after stimulation with 100 ng/ml LPS. n = at least three independent experiments. F Nlrp3 mRNA relative expression of Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS for 3 h. Data shown are pooled from three independent experiments. G Western blot protein analysis of Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS for 6 h. Data shown are representative of at least three independent experiments. Western blot was performed on whole cell lysates. H, I IL‐1β ELISA (H) and Western blot protein analysis (I) of supernatants collected from Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS and either vehicle control (Vh, DMSO) or 10 μM MCC950 for 24 h ( n = 5). Data shown in (H) are pooled from five independent replicates. J, K Caspase1 ( Casp1 ) mRNA relative expression (J) and Western blot protein analysis (K) of Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS for 3 and 6 h respectively. Data shown are pooled from three independent experiments. L IL‐1β ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS and either vehicle control (Vh, DMSO) or Ac‐YVAD‐cmk for 24 h. Data shown are pooled of five independent replicates. M IL‐1β ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 μM nigericin. Data shown are pooled of five independent replicates. N Representative picture of confocal immunofluorescence analysis of Gata6‐WT and Gata6‐KO mye pMФ stimulated with 100 ng/ml LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 μm. Data information: Data are expressed as mean ± SEM and analysis were performed using two‐way ANOVA analysis Tukey's multiple comparison post‐test unless otherwise stated. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used were goat anti‐mouse IL‐1β (catalogue number AF‐401‐NA; R&D Systems), rat anti‐human/mouse Nlrp3 (clone 768319; R&D Systems), mouse anti‐mouse caspase1 p20 (clone Casper‐1; Adipogen), rabbit anti‐mouse ASC (clone AL177, Adipogen), rabbit anti‐mouse Ptgis (catalogue number ab23668, Abcam) and mouse anti‐mouse β‐actin (clone AC‐74; Sigma).

Techniques: Expressing, Microarray, Fluorescence, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: The EMBO Journal

Article Title: Tissue‐resident macrophages actively suppress IL‐1beta release via a reactive prostanoid/IL‐10 pathway

doi: 10.15252/embj.2019103454

Figure Lengend Snippet: Western blot quantification of pro‐IL‐1β and mature IL‐1β (A), Nlrp3 (B) and caspase1 (C) of Gata6‐WT and Gata6‐KO mye pMΦ, unstimulated or stimulated overnight with 100 ng/ml LPS. Results showed are pooled from three independent experiments, normalized to Gata6‐WT unstimulated sample and are expressed as mean ± SEM. Two‐way ANOVA analysis followed by Tukey's multiple comparison post‐test was performed. * P < 0.05.

Article Snippet: The primary antibodies used were goat anti‐mouse IL‐1β (catalogue number AF‐401‐NA; R&D Systems), rat anti‐human/mouse Nlrp3 (clone 768319; R&D Systems), mouse anti‐mouse caspase1 p20 (clone Casper‐1; Adipogen), rabbit anti‐mouse ASC (clone AL177, Adipogen), rabbit anti‐mouse Ptgis (catalogue number ab23668, Abcam) and mouse anti‐mouse β‐actin (clone AC‐74; Sigma).

Techniques: Western Blot

Journal: The EMBO Journal

Article Title: Tissue‐resident macrophages actively suppress IL‐1beta release via a reactive prostanoid/IL‐10 pathway

doi: 10.15252/embj.2019103454

Figure Lengend Snippet: mRNA expression analysis of Il1b , Nlrp3 and caspase1 ( Casp1 ) of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 3 h with 100 ng/ml LPS, 10 μM beraprost, 10 ng/ml IL‐10, 5 μg/ml αIL‐10R or 5 μg/ml isotype. Data are representative of at least three independent experiments. Western blot analysis (left) and quantification (right) of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 6 h with 100 ng/ml LPS, 10 μM beraprost, 10 ng/ml IL‐10 or 5 μg/ml αIL‐10R. Data are representative of three independent experiments. Caspase1 activity analysis of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 16 h with 100 ng/ml LPS, 10 μM beraprost, 10 ng/ml IL‐10, 5 μg/ml αIL‐10R or 5 μg/ml isotype. n = 5–8 individual mice. Mean fluorescence intensity (MFI) of pro‐IL‐1β of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 3 h with 100 ng/ml LPS, 10 μM beraprost, 10 ng/ml IL‐10, 5 μg/ml αIL‐10R or 5 μg/ml isotype for 16 h or freshly isolated. n = 6–13 individual mice. Data were log‐transformed before performing statistical analysis. mRNA expression analysis of Il10 and Tnf of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 3 h with 100 ng/ml LPS, 10 μM beraprost, 10 ng/ml IL‐10 or 5 μg/ml αIL‐10R. Data are representative of at least three independent experiments. Representation of the mechanism of action of the Gata6‐PGI2‐IL‐10 active inhibitory signal on IL‐1β processing. Data information: Data are expressed as mean ± SEM, and two‐way ANOVA statistical analysis with Tukey's multiple comparison post‐test was performed. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used were goat anti‐mouse IL‐1β (catalogue number AF‐401‐NA; R&D Systems), rat anti‐human/mouse Nlrp3 (clone 768319; R&D Systems), mouse anti‐mouse caspase1 p20 (clone Casper‐1; Adipogen), rabbit anti‐mouse ASC (clone AL177, Adipogen), rabbit anti‐mouse Ptgis (catalogue number ab23668, Abcam) and mouse anti‐mouse β‐actin (clone AC‐74; Sigma).

Techniques: Expressing, Western Blot, Activity Assay, Fluorescence, Isolation, Transformation Assay