rnase treatment ssc ics  (Worthington Biochemical)


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    Name:
    Collagenase Type 2
    Description:
    Prepared to contain higher clostripain activity Suggested for bone heart liver thyroid and salivary primary cell isolation Supplied as a dialyzed lyophilized powder
    Catalog Number:
    ls004174
    Price:
    35
    Size:
    100 mg
    Source:
    Clostridium histolyticum
    Cas Number:
    9001.12.1
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    Structured Review

    Worthington Biochemical rnase treatment ssc ics
    et-1 , ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with <t>SSc-ICs</t> or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1 ; b ifn-α ; c ifn-β . * p
    Prepared to contain higher clostripain activity Suggested for bone heart liver thyroid and salivary primary cell isolation Supplied as a dialyzed lyophilized powder
    https://www.bioz.com/result/rnase treatment ssc ics/product/Worthington Biochemical
    Average 92 stars, based on 1332 article reviews
    Price from $9.99 to $1999.99
    rnase treatment ssc ics - by Bioz Stars, 2020-08
    92/100 stars

    Images

    1) Product Images from "Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts"

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1689-6

    et-1 , ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1 ; b ifn-α ; c ifn-β . * p
    Figure Legend Snippet: et-1 , ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1 ; b ifn-α ; c ifn-β . * p

    Techniques Used: Expressing, Concentration Assay

    Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p
    Figure Legend Snippet: Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p

    Techniques Used: Software, Western Blot

    TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p
    Figure Legend Snippet: TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p

    Techniques Used: Incubation, Inhibition

    TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p
    Figure Legend Snippet: TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

    Techniques Used: Expressing, Positive Control

    TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. *** p
    Figure Legend Snippet: TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. *** p

    Techniques Used: Incubation, Inhibition

    et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p
    Figure Legend Snippet: et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p

    Techniques Used: Expressing, Acetylene Reduction Assay

    tlr mRNA expression levels in fibroblasts stimulated with SSc-ICs or control NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a tlr2 ; b tlr3 ; c tlr4 ; d tlr9 . ** p
    Figure Legend Snippet: tlr mRNA expression levels in fibroblasts stimulated with SSc-ICs or control NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a tlr2 ; b tlr3 ; c tlr4 ; d tlr9 . ** p

    Techniques Used: Expressing, Concentration Assay

    TaqMan® Gene Expression assays against SSc-specific antigens of PEG-precipitated ICs and corresponding sera evaluated by EUROLINE-SSc profile kit. One ATA-IC and one NHS-IC presented as representative assay. CTR+, assay-positive control. a, Ro-52; b, PDGF receptor; c, Ku; d, PM-Scl75; e, PM-Scl100; f, Th/To; g, NOR90; h, Fibrillarin; i, RP155; l, RP11; m, CENP B; n, CENP A; o, Scl-70 (DNA topoisomerase I). ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects
    Figure Legend Snippet: TaqMan® Gene Expression assays against SSc-specific antigens of PEG-precipitated ICs and corresponding sera evaluated by EUROLINE-SSc profile kit. One ATA-IC and one NHS-IC presented as representative assay. CTR+, assay-positive control. a, Ro-52; b, PDGF receptor; c, Ku; d, PM-Scl75; e, PM-Scl100; f, Th/To; g, NOR90; h, Fibrillarin; i, RP155; l, RP11; m, CENP B; n, CENP A; o, Scl-70 (DNA topoisomerase I). ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects

    Techniques Used: Expressing, Positive Control

    IL-6 and IL-8 in culture supernatants from dcSSc fibroblasts incubated with SSc-ICs or NHS-ICs. dcSSc fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8. * p
    Figure Legend Snippet: IL-6 and IL-8 in culture supernatants from dcSSc fibroblasts incubated with SSc-ICs or NHS-ICs. dcSSc fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8. * p

    Techniques Used: Incubation, Concentration Assay

    Dose–response dilution curve for ICAM-1 expression on fibroblast cell surface. Fibroblasts exposed to serial two-fold dilutions (from 1:2 to 1:64) of SSc-ICs and NHS-ICs, and ICAM-1 evaluated by cell ELISA. anti-Th/To anti-Th/To antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects, OD optical density
    Figure Legend Snippet: Dose–response dilution curve for ICAM-1 expression on fibroblast cell surface. Fibroblasts exposed to serial two-fold dilutions (from 1:2 to 1:64) of SSc-ICs and NHS-ICs, and ICAM-1 evaluated by cell ELISA. anti-Th/To anti-Th/To antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects, OD optical density

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated ( a ) NFκB and ( b ) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). * p
    Figure Legend Snippet: Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated ( a ) NFκB and ( b ) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). * p

    Techniques Used: Western Blot, Inhibition

    IL-6, IL-8, MMP-2 and MCP-1 levels in culture supernatants from fibroblasts incubated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8; c MMP-2; d MCP-1. ** p
    Figure Legend Snippet: IL-6, IL-8, MMP-2 and MCP-1 levels in culture supernatants from fibroblasts incubated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8; c MMP-2; d MCP-1. ** p

    Techniques Used: Incubation, Concentration Assay

    ICAM-1 expression on fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. *** p
    Figure Legend Snippet: ICAM-1 expression on fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. *** p

    Techniques Used: Expressing, Concentration Assay

    2) Product Images from "Ventricular–subventricular zone fractones are speckled basement membranes that function as a neural stem cell niche"

    Article Title: Ventricular–subventricular zone fractones are speckled basement membranes that function as a neural stem cell niche

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E18-05-0286

    Fractones are visible as BM speckles in whole-mount V-SVZ immunostaining. (A) Whole-mount images of a mouse V-SVZ stained with an anti-panLM antibody. Left, image from the ventricle. Right, three-dimensional reconstruction of the left panel. See also Supplemental Movie S1. (B) Whole-mount V-SVZs were labeled with antibodies against individual BM proteins (red) together with anti-panLM or anti-LMγ1 antibodies (green). Each panel shows a merged image (top) and higher-magnification images (bottom) of the two channels in the boxed area. Asterisks, blood vessel BMs; arrowheads, speckled BMs. Scale bars, 10 μm.
    Figure Legend Snippet: Fractones are visible as BM speckles in whole-mount V-SVZ immunostaining. (A) Whole-mount images of a mouse V-SVZ stained with an anti-panLM antibody. Left, image from the ventricle. Right, three-dimensional reconstruction of the left panel. See also Supplemental Movie S1. (B) Whole-mount V-SVZs were labeled with antibodies against individual BM proteins (red) together with anti-panLM or anti-LMγ1 antibodies (green). Each panel shows a merged image (top) and higher-magnification images (bottom) of the two channels in the boxed area. Asterisks, blood vessel BMs; arrowheads, speckled BMs. Scale bars, 10 μm.

    Techniques Used: Immunostaining, Staining, Labeling

    3) Product Images from "Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues"

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39498-3

    ( A – E ) Endpoint PCR indicating the location on the gel of Lepr flox (1369 bp) and the allele resulting from Cre-induced excision, Lepr Δ17 (952 bp), in white adipose tissue (WAT), brown adipose tissue (BAT), adipocytes isolated from WAT, and non-adipose tissues in male and female AdipoqCre + Lepr flox/flox (+) and AdipoqCre − Lepr flox/flox (−) mice. Results in ( A – E ) are from 6 separate gels and the full-length blots/gels are presented in Supplementary Fig. S1 . In A, results are from one single gel; in B, results are from the second gel that has been cropped and the white space indicates cropping; in C, results are from the third gel; in D and E, results are from the fourth gel that has been cropped and the white space indicates cropping, and from the fifth and sixth gels. ( F – I ) Extent of Lepr recombination in AdipoqCre + Lepr flox/flox and AdipoqCre + Lepr loxTB/loxTB mice determined by qPCR. pg, perigonadal; sc, subcutaneous; HY, hypothalamus; L, liver; SM, skeletal muscle (gastrocnemius); HE, heart; P, pancreas; D, duodenum mucosa; J, jejunum mucosa; I, ileum mucosa; C, colon mucosa; S, stomach.
    Figure Legend Snippet: ( A – E ) Endpoint PCR indicating the location on the gel of Lepr flox (1369 bp) and the allele resulting from Cre-induced excision, Lepr Δ17 (952 bp), in white adipose tissue (WAT), brown adipose tissue (BAT), adipocytes isolated from WAT, and non-adipose tissues in male and female AdipoqCre + Lepr flox/flox (+) and AdipoqCre − Lepr flox/flox (−) mice. Results in ( A – E ) are from 6 separate gels and the full-length blots/gels are presented in Supplementary Fig. S1 . In A, results are from one single gel; in B, results are from the second gel that has been cropped and the white space indicates cropping; in C, results are from the third gel; in D and E, results are from the fourth gel that has been cropped and the white space indicates cropping, and from the fifth and sixth gels. ( F – I ) Extent of Lepr recombination in AdipoqCre + Lepr flox/flox and AdipoqCre + Lepr loxTB/loxTB mice determined by qPCR. pg, perigonadal; sc, subcutaneous; HY, hypothalamus; L, liver; SM, skeletal muscle (gastrocnemius); HE, heart; P, pancreas; D, duodenum mucosa; J, jejunum mucosa; I, ileum mucosa; C, colon mucosa; S, stomach.

    Techniques Used: Polymerase Chain Reaction, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction

    4) Product Images from "NLRC4 suppresses IL-17A-mediated neutrophil-dependent host defense through upregulation of IL-18 and induction of necroptosis during Gram-positive pneumonia"

    Article Title: NLRC4 suppresses IL-17A-mediated neutrophil-dependent host defense through upregulation of IL-18 and induction of necroptosis during Gram-positive pneumonia

    Journal: Mucosal immunology

    doi: 10.1038/s41385-018-0088-2

    NLRC4 deficiency augments IL-17A + γδ T cells and neutrophils to regulate neutrophil influx. (a-d) Flow cytometric analysis of the lungs from WT and Nlrc4 −/− mice either uninfected or intratracheally infected with 5 × 10 7 CFU of S. aureus. (a) FACS plots of CD3 + γδ-TCR + cells (larger) and IL-17A + CD3 + γδ-TCR + cells (smaller) at 6 and 24 hpi. Mice receiving PBS were sacrificed at 24-hour time points. (b) The percentage of total γδ T cells, (c) IL-17A producing γδ T cells, and (d) IL-17A producing CD11b + Ly6G-1 + neutrophils enumerated by flow cytometry in lungs at 6 and 24 hpi. (n= 5 mice / pneumonia group, n= 3 mice / control group). (e-g) WT and Nlrc4 −/− mice were inoculated intratracheally with 5 × 10 7 CFU of S. aureus. Nlrc4 −/− mice received an intraperitoneal injection of anti-γδ-TCR ab (GL-3) or IgG at 12 hours prior to infection. (e) Level of IL-17A, (f) number of neutrophils, and (g) bacterial burden in BALF was determined at 24 hpi. (n= 5 mice / group) (h) The CD3 + γδ-TCR + cells were sorted from the spleen of the uninfected wild-type and Nlrc4 −/− mice and incubated with S. aureus (MOI 10) for 18 hours in the presence or absence of IL-23 (40 ng/ml). (h) IL-17A was measured in the supernatant. (i and j) WT and Nlrc4 −/− mice were inoculated intratracheally with 5 × 10 7 CFU of S. aureus or PBS. These mice received a single injection of BrdU (1mg/mouse) intraperitoneally 1-hour prior to infection. (i) FACS plot showing BrdU + CD3 + γδ-TCR + cells and (j) their percentage in the lungs of mice 18 hpi. (n= 5 mice /pneumonia group, n=3 mice/control group). Data from a representative experiment are shown. All experiments were performed three times. In vitro experiments have at least four biological replicates. Statistical significance was determined by unpaired t-test (b-d) and one-way ANOVA (followed by Bonferroni’s post hoc comparisons) (e-g) * p
    Figure Legend Snippet: NLRC4 deficiency augments IL-17A + γδ T cells and neutrophils to regulate neutrophil influx. (a-d) Flow cytometric analysis of the lungs from WT and Nlrc4 −/− mice either uninfected or intratracheally infected with 5 × 10 7 CFU of S. aureus. (a) FACS plots of CD3 + γδ-TCR + cells (larger) and IL-17A + CD3 + γδ-TCR + cells (smaller) at 6 and 24 hpi. Mice receiving PBS were sacrificed at 24-hour time points. (b) The percentage of total γδ T cells, (c) IL-17A producing γδ T cells, and (d) IL-17A producing CD11b + Ly6G-1 + neutrophils enumerated by flow cytometry in lungs at 6 and 24 hpi. (n= 5 mice / pneumonia group, n= 3 mice / control group). (e-g) WT and Nlrc4 −/− mice were inoculated intratracheally with 5 × 10 7 CFU of S. aureus. Nlrc4 −/− mice received an intraperitoneal injection of anti-γδ-TCR ab (GL-3) or IgG at 12 hours prior to infection. (e) Level of IL-17A, (f) number of neutrophils, and (g) bacterial burden in BALF was determined at 24 hpi. (n= 5 mice / group) (h) The CD3 + γδ-TCR + cells were sorted from the spleen of the uninfected wild-type and Nlrc4 −/− mice and incubated with S. aureus (MOI 10) for 18 hours in the presence or absence of IL-23 (40 ng/ml). (h) IL-17A was measured in the supernatant. (i and j) WT and Nlrc4 −/− mice were inoculated intratracheally with 5 × 10 7 CFU of S. aureus or PBS. These mice received a single injection of BrdU (1mg/mouse) intraperitoneally 1-hour prior to infection. (i) FACS plot showing BrdU + CD3 + γδ-TCR + cells and (j) their percentage in the lungs of mice 18 hpi. (n= 5 mice /pneumonia group, n=3 mice/control group). Data from a representative experiment are shown. All experiments were performed three times. In vitro experiments have at least four biological replicates. Statistical significance was determined by unpaired t-test (b-d) and one-way ANOVA (followed by Bonferroni’s post hoc comparisons) (e-g) * p

    Techniques Used: Flow Cytometry, Mouse Assay, Infection, FACS, Cytometry, Injection, Incubation, In Vitro

    NLRC4-driven necroptosis and IL-18 suppress 17A-dependent neutrophil recruitment by limiting γδ T cell expansion. (a) WT and Nlrc4 −/− mice were treated with Necrostatin-1 or DMSO intraperitoneally 12 hours prior to infection with S. aureus (5 ×10 7 CFU/mouse). RFU of LDH release in BALF was measured at 24 hpi. (n= 5 mice /group). (b-g) WT, Mlkl −/− and Nlrc4 −/− mice were infected with S. aureus (5 ×10 7 CFU/mouse) intratracheally with administration of GW806742X (MLKL inhibitor) or DMSO intraperitoneally 1 hour prior. BALF and lungs were harvested at 24 hpi. (b) The percentage of total γδ T cells and (c) IL-17A producing γδ T cells in the lungs were determined by flow cytometry. (d) IL-17A level, (e) number of neutrophils, and (f) bacterial burden in BALF, and (g) lungs were quantitated. Each symbol represents a single mouse. (n= 4-6 mice /group). (h-m) WT, IL-18 −/− , Il-1β −/− , and Nlrc4 −/− mice were infected with S. aureus (5 ×10 7 CFU/mouse) intratracheally. At 24 hpi, the lungs were harvested and processed for flow cytometric analysis. (h) The percentage of total γδ T cells and (i) IL-17A producing γδ T cells in the lungs of WT, IL-18, −/− , and Nlrc4 −/− mice receiving recombinant murine IL-18 (1μg/mouse) or PBS 1 hpi were enumerated. (j) The percentage of total γδ T cells and (k) IL-17A producing γδ T cells in the lungs of WT, Il-1β, −/− and Nlrc4 −/− mice. (l) The number of neutrophils in BALF and (m) bacterial burden in the lungs at 24 hpi. Each symbol represents a single mouse. (n= 4-6/group). Data from a representative experiment are shown. All experiments were performed three times. Statistical significance was determined by one-way ANOVA (followed by Bonferroni’s post hoc comparisons) in all experiments. * p
    Figure Legend Snippet: NLRC4-driven necroptosis and IL-18 suppress 17A-dependent neutrophil recruitment by limiting γδ T cell expansion. (a) WT and Nlrc4 −/− mice were treated with Necrostatin-1 or DMSO intraperitoneally 12 hours prior to infection with S. aureus (5 ×10 7 CFU/mouse). RFU of LDH release in BALF was measured at 24 hpi. (n= 5 mice /group). (b-g) WT, Mlkl −/− and Nlrc4 −/− mice were infected with S. aureus (5 ×10 7 CFU/mouse) intratracheally with administration of GW806742X (MLKL inhibitor) or DMSO intraperitoneally 1 hour prior. BALF and lungs were harvested at 24 hpi. (b) The percentage of total γδ T cells and (c) IL-17A producing γδ T cells in the lungs were determined by flow cytometry. (d) IL-17A level, (e) number of neutrophils, and (f) bacterial burden in BALF, and (g) lungs were quantitated. Each symbol represents a single mouse. (n= 4-6 mice /group). (h-m) WT, IL-18 −/− , Il-1β −/− , and Nlrc4 −/− mice were infected with S. aureus (5 ×10 7 CFU/mouse) intratracheally. At 24 hpi, the lungs were harvested and processed for flow cytometric analysis. (h) The percentage of total γδ T cells and (i) IL-17A producing γδ T cells in the lungs of WT, IL-18, −/− , and Nlrc4 −/− mice receiving recombinant murine IL-18 (1μg/mouse) or PBS 1 hpi were enumerated. (j) The percentage of total γδ T cells and (k) IL-17A producing γδ T cells in the lungs of WT, Il-1β, −/− and Nlrc4 −/− mice. (l) The number of neutrophils in BALF and (m) bacterial burden in the lungs at 24 hpi. Each symbol represents a single mouse. (n= 4-6/group). Data from a representative experiment are shown. All experiments were performed three times. Statistical significance was determined by one-way ANOVA (followed by Bonferroni’s post hoc comparisons) in all experiments. * p

    Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, Recombinant

    5) Product Images from "Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector"

    Article Title: Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00415

    A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .
    Figure Legend Snippet: A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .

    Techniques Used: Injection, Expressing, Labeling, Marker

    Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.
    Figure Legend Snippet: Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.

    Techniques Used: Injection, Expressing, Labeling, Staining

    6) Product Images from "Heme oxygenase-1 induction contributes to renoprotection by G-CSF during rhabdomyolysis-associated acute kidney injury"

    Article Title: Heme oxygenase-1 induction contributes to renoprotection by G-CSF during rhabdomyolysis-associated acute kidney injury

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00438.2010

    HO-1 induction by G-CSF in mouse kidneys. A : immunoblot analysis of HO-1 in mouse kidney cortex. C57BL/6 mice were treated with 200 mg·kg −1 ·day −1 G-CSF or saline for 5 days. Renal cortical tissues were collected and homogenized for immunoblot analysis of HO-1 and β-actin. B : immunohistochemical staining of HO-1 in mouse kidneys. C57BL/6 mice were treated with 200 mg·kg −1 ·day −1 G-CSF or saline for 5 days to collect kidney tissues for fixation with paraformaldehyde for immunohistochemistry. The bottom micrographs are magnified images of the boxed areas in the top panels. *, Glomeruli. The results show that HO-1 is mainly induced by G-CSF renal tubules.
    Figure Legend Snippet: HO-1 induction by G-CSF in mouse kidneys. A : immunoblot analysis of HO-1 in mouse kidney cortex. C57BL/6 mice were treated with 200 mg·kg −1 ·day −1 G-CSF or saline for 5 days. Renal cortical tissues were collected and homogenized for immunoblot analysis of HO-1 and β-actin. B : immunohistochemical staining of HO-1 in mouse kidneys. C57BL/6 mice were treated with 200 mg·kg −1 ·day −1 G-CSF or saline for 5 days to collect kidney tissues for fixation with paraformaldehyde for immunohistochemistry. The bottom micrographs are magnified images of the boxed areas in the top panels. *, Glomeruli. The results show that HO-1 is mainly induced by G-CSF renal tubules.

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    Effects of G-CSF on animal survival following glycerol injection. C57BL/6 mice (male, 8–10 wk) were divided into 2 groups. One group was pretreated for 5 days with 200 mg·kg −1 ·day −1 G-CSF ( n = 7), and the other group was pretreated with saline ( n = 14). Both groups of animals were then injected with 7.5 ml/kg 50% glycerol in hindleg muscles. Animal death and survival in these 2 groups were recorded for 10 days following glycerol injection. The results show a beneficial effect of G-CSF on animal survival following glycerol-induced rhabdomyolysis in mice. *Significant difference between the survival rates of the 2 groups ( P
    Figure Legend Snippet: Effects of G-CSF on animal survival following glycerol injection. C57BL/6 mice (male, 8–10 wk) were divided into 2 groups. One group was pretreated for 5 days with 200 mg·kg −1 ·day −1 G-CSF ( n = 7), and the other group was pretreated with saline ( n = 14). Both groups of animals were then injected with 7.5 ml/kg 50% glycerol in hindleg muscles. Animal death and survival in these 2 groups were recorded for 10 days following glycerol injection. The results show a beneficial effect of G-CSF on animal survival following glycerol-induced rhabdomyolysis in mice. *Significant difference between the survival rates of the 2 groups ( P

    Techniques Used: Injection, Mouse Assay

    Effects of granulocyte colony-stimulating factor (G-CSF) on glycerol-induced kidney injury. C57BL/6 mice (male, 8–10 wk) were pretreated for 5 days with 200 mg·kg −1 ·day −1 G-CSF or saline. The animals were then injected with 7.5 ml/kg 50% glycerol in the hindleg muscles. A : blood urea nitrogen (BUN) levels. Blood samples were collected just before glycerol injection or 1–2 days after glycerol injection to determine BUN. *Significantly different from day 0 ( P
    Figure Legend Snippet: Effects of granulocyte colony-stimulating factor (G-CSF) on glycerol-induced kidney injury. C57BL/6 mice (male, 8–10 wk) were pretreated for 5 days with 200 mg·kg −1 ·day −1 G-CSF or saline. The animals were then injected with 7.5 ml/kg 50% glycerol in the hindleg muscles. A : blood urea nitrogen (BUN) levels. Blood samples were collected just before glycerol injection or 1–2 days after glycerol injection to determine BUN. *Significantly different from day 0 ( P

    Techniques Used: Mouse Assay, Injection

    7) Product Images from "Lipids Cooperate with the Reovirus Membrane Penetration Peptide to Facilitate Particle Uncoating"

    Article Title: Lipids Cooperate with the Reovirus Membrane Penetration Peptide to Facilitate Particle Uncoating

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.747477

    Liposomes facilitate thermal inactivation of T3D ISVPs in a virus concentration-dependent manner. A and C , lipid-mediated thermal inactivation curves. T3D ISVPs at 2 × 10 12 particles/ml ( A ) or 2 × 10 9 particles/ml ( C ) were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4 °C was determined by plaque assay. Data are presented as mean ± S.D., *, p ≤ 0.01 ( n = 3 independent replicates for each reaction condition). B and D , liposome-mediated ISVP* formation. T3D ISVPs at 2 × 10 12 particles/ml ( B ) or 2 × 10 9 particles/ml ( D ) were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. In panel B , the gels were Coomassie Brilliant Blue-stained. In panel D , the gels were analyzed for the presence of the μ1 δ fragment by Western blotting ( n = 3 independent replicates for each reaction condition, one representative experiment is shown).
    Figure Legend Snippet: Liposomes facilitate thermal inactivation of T3D ISVPs in a virus concentration-dependent manner. A and C , lipid-mediated thermal inactivation curves. T3D ISVPs at 2 × 10 12 particles/ml ( A ) or 2 × 10 9 particles/ml ( C ) were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4 °C was determined by plaque assay. Data are presented as mean ± S.D., *, p ≤ 0.01 ( n = 3 independent replicates for each reaction condition). B and D , liposome-mediated ISVP* formation. T3D ISVPs at 2 × 10 12 particles/ml ( B ) or 2 × 10 9 particles/ml ( D ) were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. In panel B , the gels were Coomassie Brilliant Blue-stained. In panel D , the gels were analyzed for the presence of the μ1 δ fragment by Western blotting ( n = 3 independent replicates for each reaction condition, one representative experiment is shown).

    Techniques Used: Concentration Assay, Incubation, Infection, Plaque Assay, SDS Page, Staining, Western Blot

    The supernatant of preconverted ISVP*s cooperates with liposomes to facilitate thermal inactivation of T3D ISVPs. A and B , generation of ISVP* supernatant. Input T3D ISVPs at 2 × 10 12 particles/ml were incubated at 52 °C for 5 min. The heat-inactivated virus ( No spin ) was centrifuged to pellet particles. The supernatant ( Spin ) was immediately transferred to tubes containing target T3D ISVPs for thermal inactivation reactions. Aliquots of the no spin and spin reactions were analyzed for residual infectivity by plaque assay ( A ) and for the presence of the μ1 δ fragment by Western blotting ( B ). In panel A , data are presented as mean ± S.D., *, p ≤ 0.01 ( n = 3 independent preparations of ISVP* supernatant were analyzed). C , ISVP* supernatant- and liposome-mediated thermal inactivation curves. T3D ISVPs at 2 × 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and ISVP* supernatant for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4 °C was determined by plaque assay. Data are presented as mean ± S.D., *, p ≤ 0.01 ( n = 3 independent replicates for each reaction condition). D , ISVP* supernatant- and liposome-mediated ISVP* formation. T3D ISVPs at 2 × 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and ISVP* supernatant for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were analyzed for the presence of the μ1 δ fragment by Western blotting ( n = 3 independent replicates for each reaction condition, one representative experiment is shown).
    Figure Legend Snippet: The supernatant of preconverted ISVP*s cooperates with liposomes to facilitate thermal inactivation of T3D ISVPs. A and B , generation of ISVP* supernatant. Input T3D ISVPs at 2 × 10 12 particles/ml were incubated at 52 °C for 5 min. The heat-inactivated virus ( No spin ) was centrifuged to pellet particles. The supernatant ( Spin ) was immediately transferred to tubes containing target T3D ISVPs for thermal inactivation reactions. Aliquots of the no spin and spin reactions were analyzed for residual infectivity by plaque assay ( A ) and for the presence of the μ1 δ fragment by Western blotting ( B ). In panel A , data are presented as mean ± S.D., *, p ≤ 0.01 ( n = 3 independent preparations of ISVP* supernatant were analyzed). C , ISVP* supernatant- and liposome-mediated thermal inactivation curves. T3D ISVPs at 2 × 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and ISVP* supernatant for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4 °C was determined by plaque assay. Data are presented as mean ± S.D., *, p ≤ 0.01 ( n = 3 independent replicates for each reaction condition). D , ISVP* supernatant- and liposome-mediated ISVP* formation. T3D ISVPs at 2 × 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and ISVP* supernatant for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were analyzed for the presence of the μ1 δ fragment by Western blotting ( n = 3 independent replicates for each reaction condition, one representative experiment is shown).

    Techniques Used: Incubation, Infection, Plaque Assay, Western Blot, SDS Page

    8) Product Images from "RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer"

    Article Title: RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer

    Journal: Genes & Development

    doi: 10.1101/gad.304162.117

    RANK is expressed in human lung tumors and controls growth of human lung cancer. ( A ) Representative RANK IHC in primary human NSCLC lung adenocarcinomas. Bars, 100 µm. ( B ) Cross-correlation matrixes to compare RANK protein expression (determined by IHC) in human lung tumors with KRAS mutational status and smoking. n = 364, ”Uppsala” cohort with early stage treatment-naïve resected lung cancer, including squamous cell carcinoma, adenocarcinoma, SCLC, and large-cell carcinoma. P -values are indicated, calculated using the Fisher's exact test. ( C ) Prediction of overall survival probability in the human Affymetrix lung adenocarcinoma data set stratified for high (red lines) and low (black lines) RANK, RANKL, OPG, and LGR4 mRNA expression based on the best fit algorithm. Data were obtained using KM plotter. P -values (log rank test) are indicated. ( D ) RANK IHC in a patient-derived KRAS mutant adenocarcinoma used for xenograft experiments. Bars, 100 µm. ( E ) Growth of a patient-derived lung adenocarcinoma xenograft (PDX). PDX tumor fragments were implanted subcutaneously in the right flanks of 6- to 8-wk-old female NSG (NOD- scid IL2Rγ null ) mice. At day 45 (when tumors had reached ∼200 mm 3 ), mice were treated with 10 mg/kg OPG-Fc or PBS subcutaneously twice per week, and tumor volumes were measured twice per week using digital calipers. OPG-Fc treatment significantly reduced tumor burden. P = 0.024, repeated measures analysis of variance (RMANOVA) followed by Dunnett's post-hoc test. Data represent the mean ± SEM for each group. n = 10 per group. ( F ) Proliferation of PDX tumor cells in PBS and 10 mg/kg OPG-Fc-treated mice was determined using Ki67 immunostaining, analyzed at the termination of the experiment for ethical reasons. Data are shown as mean percentage of Ki67 + cells (±SEM) among tumor cells, comparing the two groups. n = 10 per group. P = 0.0143, unpaired t -test.
    Figure Legend Snippet: RANK is expressed in human lung tumors and controls growth of human lung cancer. ( A ) Representative RANK IHC in primary human NSCLC lung adenocarcinomas. Bars, 100 µm. ( B ) Cross-correlation matrixes to compare RANK protein expression (determined by IHC) in human lung tumors with KRAS mutational status and smoking. n = 364, ”Uppsala” cohort with early stage treatment-naïve resected lung cancer, including squamous cell carcinoma, adenocarcinoma, SCLC, and large-cell carcinoma. P -values are indicated, calculated using the Fisher's exact test. ( C ) Prediction of overall survival probability in the human Affymetrix lung adenocarcinoma data set stratified for high (red lines) and low (black lines) RANK, RANKL, OPG, and LGR4 mRNA expression based on the best fit algorithm. Data were obtained using KM plotter. P -values (log rank test) are indicated. ( D ) RANK IHC in a patient-derived KRAS mutant adenocarcinoma used for xenograft experiments. Bars, 100 µm. ( E ) Growth of a patient-derived lung adenocarcinoma xenograft (PDX). PDX tumor fragments were implanted subcutaneously in the right flanks of 6- to 8-wk-old female NSG (NOD- scid IL2Rγ null ) mice. At day 45 (when tumors had reached ∼200 mm 3 ), mice were treated with 10 mg/kg OPG-Fc or PBS subcutaneously twice per week, and tumor volumes were measured twice per week using digital calipers. OPG-Fc treatment significantly reduced tumor burden. P = 0.024, repeated measures analysis of variance (RMANOVA) followed by Dunnett's post-hoc test. Data represent the mean ± SEM for each group. n = 10 per group. ( F ) Proliferation of PDX tumor cells in PBS and 10 mg/kg OPG-Fc-treated mice was determined using Ki67 immunostaining, analyzed at the termination of the experiment for ethical reasons. Data are shown as mean percentage of Ki67 + cells (±SEM) among tumor cells, comparing the two groups. n = 10 per group. P = 0.0143, unpaired t -test.

    Techniques Used: Immunohistochemistry, Expressing, Derivative Assay, Mutagenesis, Mouse Assay, Immunostaining

    9) Product Images from "DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides"

    Article Title: DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051905

    Mechanical strength, anti-disruptive properties and nanomechanical adhesive characteristics recorded using force-separation curves of the M. xanthus starvation biofilm matrix with or without eDNA. In panel A, DK1622 biofilms without DNase I (black bar) and with DNase I (grey bar) were established over a 24 hr time period in MOPS buffer and biomass was measured as crystal violet optical density (No-treatment control). The changes of biomass in these two kinds of biofilms after sonication or SDS treatment, respectively, are also shown. In panel B, representative force curves measured by AFM on DK1622 24 hr starvation biofilm matrix (curve I), matrix treated with DNase I (curve II) and bare portion of the substrate after the tip was used (curve III). Force-separation curves were recorded as “approach” (blue) and “retraction” (red) curves.
    Figure Legend Snippet: Mechanical strength, anti-disruptive properties and nanomechanical adhesive characteristics recorded using force-separation curves of the M. xanthus starvation biofilm matrix with or without eDNA. In panel A, DK1622 biofilms without DNase I (black bar) and with DNase I (grey bar) were established over a 24 hr time period in MOPS buffer and biomass was measured as crystal violet optical density (No-treatment control). The changes of biomass in these two kinds of biofilms after sonication or SDS treatment, respectively, are also shown. In panel B, representative force curves measured by AFM on DK1622 24 hr starvation biofilm matrix (curve I), matrix treated with DNase I (curve II) and bare portion of the substrate after the tip was used (curve III). Force-separation curves were recorded as “approach” (blue) and “retraction” (red) curves.

    Techniques Used: Sonication

    eDNA colocalized with EPS within M. xanthus starvation biofilm and fruiting body structures. Panel A, DK1622 starvation biofilm formed in MOPS buffer for 24 hr labeled with STYOX orange (red), Alexa 633-conjugated WGA (blue) and SYTO 9 (green). Panel B, DK1622 24 hr fruiting body structures with STYOX orange, Alexa 633-conjugated WGA and SYTO 9. Images in panel A were taken with a 40× objective using CLSM, and images in panel B were taken with a 63× objective. The bars in panels A and B represent 50 µM. Panel C showed the quantitative colocalization analysis results of STYOX orange (eDNA) and Alexa 633-WGA (EPS) signals from submerged 24 hr starvation biofilms (left) and 24 hr fruiting bodies (right). The PCC represents Pearson’s correlation coefficient, MOC represents overlap coefficients according to Manders, M1 represents colocalization coefficient M1 (fraction of eDNA overlapping EPS), M2 represents colocalization coefficient M2 (fraction of EPS overlapping eDNA), and ICQ represents intensity correlation quotient. Mean ± SD is plotted.
    Figure Legend Snippet: eDNA colocalized with EPS within M. xanthus starvation biofilm and fruiting body structures. Panel A, DK1622 starvation biofilm formed in MOPS buffer for 24 hr labeled with STYOX orange (red), Alexa 633-conjugated WGA (blue) and SYTO 9 (green). Panel B, DK1622 24 hr fruiting body structures with STYOX orange, Alexa 633-conjugated WGA and SYTO 9. Images in panel A were taken with a 40× objective using CLSM, and images in panel B were taken with a 63× objective. The bars in panels A and B represent 50 µM. Panel C showed the quantitative colocalization analysis results of STYOX orange (eDNA) and Alexa 633-WGA (EPS) signals from submerged 24 hr starvation biofilms (left) and 24 hr fruiting bodies (right). The PCC represents Pearson’s correlation coefficient, MOC represents overlap coefficients according to Manders, M1 represents colocalization coefficient M1 (fraction of eDNA overlapping EPS), M2 represents colocalization coefficient M2 (fraction of EPS overlapping eDNA), and ICQ represents intensity correlation quotient. Mean ± SD is plotted.

    Techniques Used: Labeling, Whole Genome Amplification, Confocal Laser Scanning Microscopy, Periodic Counter-current Chromatography

    eDNA in M. xanthus non-developmental starvation biofilms. M. xanthus DK1622 biofilm structures formed in MOPS buffer at 24 hr (panels A and B) and DNase I treated biofilm (panels C and D) were counterstained with SYTOX orange (red) and SYTO9 (green). Panels A and C are the single channel images (SYTOX orange), and panels B and D are the overlaid images. M. xanthus DK10547 with a GFP label (green) formed starvation biofilms (panels E and F) in MOPs buffer at 24 hr was counterstained with SYTOX orange (red) and FM 4-64 (blue). Panel E is the single channel image (FM 4-64), and panel F is the overlaid image. The bars represent 50 µm in panels A–D and 10 µm in panels E and F.
    Figure Legend Snippet: eDNA in M. xanthus non-developmental starvation biofilms. M. xanthus DK1622 biofilm structures formed in MOPS buffer at 24 hr (panels A and B) and DNase I treated biofilm (panels C and D) were counterstained with SYTOX orange (red) and SYTO9 (green). Panels A and C are the single channel images (SYTOX orange), and panels B and D are the overlaid images. M. xanthus DK10547 with a GFP label (green) formed starvation biofilms (panels E and F) in MOPs buffer at 24 hr was counterstained with SYTOX orange (red) and FM 4-64 (blue). Panel E is the single channel image (FM 4-64), and panel F is the overlaid image. The bars represent 50 µm in panels A–D and 10 µm in panels E and F.

    Techniques Used:

    10) Product Images from "Apoptosis Repressor with a CARD Domain (ARC) Restrains Bax-Mediated Pathogenesis in Dystrophic Skeletal Muscle"

    Article Title: Apoptosis Repressor with a CARD Domain (ARC) Restrains Bax-Mediated Pathogenesis in Dystrophic Skeletal Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082053

    Arc deficiency increases Bax expression and cell death. A, Western blot for Bak and Bax from quadriceps lysates of Nol3 -/- , Sgcd -/- , and Nol3 -/- Sgcd -/- mice. (β-tubulin serves as a loading control). B, Western blot for Bax and Bcl-X L from mitochondrial protein fractions isolated from pooled hindlimb muscles of WT, Nol3 -/- , Sgcd -/- , and Nol3 -/- Sgcd -/- mice. (voltage-dependent anion channel (VDAC) serves as a mitochondrial protein loading control). C, Western blotting for Arc from lysates derived from WT and Bak -/- Bak1 -/- SV40 transformed MEFs. Skeletal muscle lysates were included to show the enrichment of Arc in terminally differentiated cell types, while GAPDH serves as a protein loading control. D, Western blot for Bax and Arc in SV40 transformed MEFs infected with lentivirus expressing scrambled shRNA (con) or 3 different Bax-directed shRNAs. (GAPDH serves as a loading control). E, Western blot for Arc in SV40 transformed MEFs infected with lentivirus expressing either a scrambled shRNA or one of the Bax shRNAs and treated with proteosomal inhibitors Bortezomib or MG-132. WT MEFs are a control for normal endogenous Arc expression and GAPDH serves as a loading control. F, Western blot for Arc in SV40 transformed MEFs infected with a lentivirus expressing either a scrambled shRNA (con.) or shRNA directed against Arc. Western blots presented are quantified and statistically analyzed in Figure S3A-G . G, Quantification of dead cells by flow cytometry sorting for Annexin and PI positivity in the experimental groups shown, treated or untreated with staurosporin for 12 hours. *P
    Figure Legend Snippet: Arc deficiency increases Bax expression and cell death. A, Western blot for Bak and Bax from quadriceps lysates of Nol3 -/- , Sgcd -/- , and Nol3 -/- Sgcd -/- mice. (β-tubulin serves as a loading control). B, Western blot for Bax and Bcl-X L from mitochondrial protein fractions isolated from pooled hindlimb muscles of WT, Nol3 -/- , Sgcd -/- , and Nol3 -/- Sgcd -/- mice. (voltage-dependent anion channel (VDAC) serves as a mitochondrial protein loading control). C, Western blotting for Arc from lysates derived from WT and Bak -/- Bak1 -/- SV40 transformed MEFs. Skeletal muscle lysates were included to show the enrichment of Arc in terminally differentiated cell types, while GAPDH serves as a protein loading control. D, Western blot for Bax and Arc in SV40 transformed MEFs infected with lentivirus expressing scrambled shRNA (con) or 3 different Bax-directed shRNAs. (GAPDH serves as a loading control). E, Western blot for Arc in SV40 transformed MEFs infected with lentivirus expressing either a scrambled shRNA or one of the Bax shRNAs and treated with proteosomal inhibitors Bortezomib or MG-132. WT MEFs are a control for normal endogenous Arc expression and GAPDH serves as a loading control. F, Western blot for Arc in SV40 transformed MEFs infected with a lentivirus expressing either a scrambled shRNA (con.) or shRNA directed against Arc. Western blots presented are quantified and statistically analyzed in Figure S3A-G . G, Quantification of dead cells by flow cytometry sorting for Annexin and PI positivity in the experimental groups shown, treated or untreated with staurosporin for 12 hours. *P

    Techniques Used: Expressing, Western Blot, Mouse Assay, Isolation, Derivative Assay, Transformation Assay, Infection, shRNA, Flow Cytometry, Cytometry

    11) Product Images from "Circulating tumour necrosis factor-? bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response"

    Article Title: Circulating tumour necrosis factor-? bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar1465

    Pattern of plasma tumour necrosis factor (TNF) bioactivity in rheumatoid arthritis (RA) patients and healthy individuals. Using the ability of TNF-α to stimulate RA synoviocytes, TNF-α (5 ng/ml) was combined with increasing concentrations of plasma from RA patients (0–20%). (a) Four RA plasma samples were obtained before infliximab therapy. (b) RA plasma samples ( n = 2) were used at 10% dilution. IL-6 production was measured by ELISA in 48-hour supernatants.
    Figure Legend Snippet: Pattern of plasma tumour necrosis factor (TNF) bioactivity in rheumatoid arthritis (RA) patients and healthy individuals. Using the ability of TNF-α to stimulate RA synoviocytes, TNF-α (5 ng/ml) was combined with increasing concentrations of plasma from RA patients (0–20%). (a) Four RA plasma samples were obtained before infliximab therapy. (b) RA plasma samples ( n = 2) were used at 10% dilution. IL-6 production was measured by ELISA in 48-hour supernatants.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Link between changes in IL-6 and osteoprotegerin (OPG) production by rheumatoid arthritis (RA) synoviocytes and clinical response. Changes, expressed as ΔIL-6 and ΔOPG production by RA synoviocytes before and 4 hours after the first infliximab infusion, are separated according to the American College of Rheumatology (ACR) clinical response (good or poor) at 54 weeks ( n = 20). A good clinical response was defined as an ACR 50 response or better ( n = 12).
    Figure Legend Snippet: Link between changes in IL-6 and osteoprotegerin (OPG) production by rheumatoid arthritis (RA) synoviocytes and clinical response. Changes, expressed as ΔIL-6 and ΔOPG production by RA synoviocytes before and 4 hours after the first infliximab infusion, are separated according to the American College of Rheumatology (ACR) clinical response (good or poor) at 54 weeks ( n = 20). A good clinical response was defined as an ACR 50 response or better ( n = 12).

    Techniques Used:

    Principle of the bioassay. Rheumatoid arthritis synoviocytes (10 4 cells/well) were cultured in 96-well plates and stimulated with increasing doses of tumour necrosis factor (TNF)-α (0–100 ng/ml). Levels of (a) IL-6 and (b) osteoprotegerin (OPG) were measured in 48-hour supernatants. Infliximab at 10 μg/ml was preincubated for 1 hour with TNF-α before its addition to the culture.
    Figure Legend Snippet: Principle of the bioassay. Rheumatoid arthritis synoviocytes (10 4 cells/well) were cultured in 96-well plates and stimulated with increasing doses of tumour necrosis factor (TNF)-α (0–100 ng/ml). Levels of (a) IL-6 and (b) osteoprotegerin (OPG) were measured in 48-hour supernatants. Infliximab at 10 μg/ml was preincubated for 1 hour with TNF-α before its addition to the culture.

    Techniques Used: Cell Culture

    Link between circulating tumour necrosis factor (TNF)-α bioactivity and clinical response. Using the ability of TNF-α to stimulate rheumatoid arthritis synoviocytes, TNF-α (5 ng/ml) was combined with 20 μl plasma per well in order to increase the sensitivity of synoviocyte response. Levels of circulating TNF-α bioactivity were estimated with plasma samples obtained before infliximab therapy and separated according to American College of Rheumatology (ACR) clinical response (good or poor) at 54 weeks ( n = 42). A good clinical response was defined as an ACR 50 response or better ( n = 24).
    Figure Legend Snippet: Link between circulating tumour necrosis factor (TNF)-α bioactivity and clinical response. Using the ability of TNF-α to stimulate rheumatoid arthritis synoviocytes, TNF-α (5 ng/ml) was combined with 20 μl plasma per well in order to increase the sensitivity of synoviocyte response. Levels of circulating TNF-α bioactivity were estimated with plasma samples obtained before infliximab therapy and separated according to American College of Rheumatology (ACR) clinical response (good or poor) at 54 weeks ( n = 42). A good clinical response was defined as an ACR 50 response or better ( n = 24).

    Techniques Used:

    12) Product Images from "Tetraspanin CD151 plays a key role in skin squamous cell carcinoma"

    Article Title: Tetraspanin CD151 plays a key role in skin squamous cell carcinoma

    Journal: Oncogene

    doi: 10.1038/onc.2012.205

    CD151 affects β4 distribution and phosphorylation. A , Mouse tumor-derived cell lines were permeabilized and stained for integrin β4. B , Keratinocytes were isolated from wild type and null mice and then cultured for a few weeks before integrin β4 subunit staining. Bar = 5 µm . ( A, B ) Linear staining intensity, across the diameter of representative cells, was measured using the Image J program (from NIH). C, Human A431 cells, stably ablated for CD151, were treated with TPA (50ng/ml) or EGF (100ng/ml) for 5 min and 1 hr, and then blotted for the indicated proteins, using antibodies for β4-S1424, β4-S1356, and total β4.
    Figure Legend Snippet: CD151 affects β4 distribution and phosphorylation. A , Mouse tumor-derived cell lines were permeabilized and stained for integrin β4. B , Keratinocytes were isolated from wild type and null mice and then cultured for a few weeks before integrin β4 subunit staining. Bar = 5 µm . ( A, B ) Linear staining intensity, across the diameter of representative cells, was measured using the Image J program (from NIH). C, Human A431 cells, stably ablated for CD151, were treated with TPA (50ng/ml) or EGF (100ng/ml) for 5 min and 1 hr, and then blotted for the indicated proteins, using antibodies for β4-S1424, β4-S1356, and total β4.

    Techniques Used: Derivative Assay, Staining, Isolation, Mouse Assay, Cell Culture, Stable Transfection

    CD151 and STAT3 inhibition effects. A , Equal numbers of freshly isolated primary mouse keratinocytes were cultured ± 5 µM nifuroxazide. After 2 days, cell proliferation was assessed using the MTT assay, which measures metabolic energy (left panel) and after 3 days total cell numbers were counted (right panel). Bars represent mean +/− SD for 3 independent experiments. *, P
    Figure Legend Snippet: CD151 and STAT3 inhibition effects. A , Equal numbers of freshly isolated primary mouse keratinocytes were cultured ± 5 µM nifuroxazide. After 2 days, cell proliferation was assessed using the MTT assay, which measures metabolic energy (left panel) and after 3 days total cell numbers were counted (right panel). Bars represent mean +/− SD for 3 independent experiments. *, P

    Techniques Used: Inhibition, Isolation, Cell Culture, MTT Assay

    13) Product Images from "The bovine mammary gland expresses multiple functional isoforms of serotonin receptors"

    Article Title: The bovine mammary gland expresses multiple functional isoforms of serotonin receptors

    Journal: The Journal of Endocrinology

    doi: 10.1677/JOE-09-0187

    Caspase-3 activity in primary bovine mammary epithelial cells treated with 5-HT receptor antagonists. Mean with s.e.m . of caspase-3 activity in lactogenic cultures of BMEC treated with 1·0 μM SB224289, 1·0 μM ritanserin, 0·1 μM SB204741, 0·0001 μM SB204070, and 0·1 μM pimozide. Columns with * indicate statistical significance of * P
    Figure Legend Snippet: Caspase-3 activity in primary bovine mammary epithelial cells treated with 5-HT receptor antagonists. Mean with s.e.m . of caspase-3 activity in lactogenic cultures of BMEC treated with 1·0 μM SB224289, 1·0 μM ritanserin, 0·1 μM SB204741, 0·0001 μM SB204070, and 0·1 μM pimozide. Columns with * indicate statistical significance of * P

    Techniques Used: Activity Assay

    14) Product Images from "Wnt4 is not sufficient to induce lobuloalveolar mammary development"

    Article Title: Wnt4 is not sufficient to induce lobuloalveolar mammary development

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-9-55

    Wnt4 does not induce canonical Wnt signaling in mammary epithelial or fibroblast cells in vitro . (A) Wnt reporter assay of primary mammary epithelial cells. Primary mammary epithelial cells were transfected with superTOP-flash reporter gene, a Renilla luciferase gene (a transfection control), and various amounts of Wnt4 expression vector (0.05~2.0 μg). Luciferase activity was measured 48 hrs after transfection, and results corrected for transfection efficiency (dividing by Renilla luciferase activity). Fold induction was calculated with respect to the negative control ( LacZ -transfected cells). A Wnt1 expression vector was used as a positive control. (B) Wnt reporter assay of immortalized mammary fibroblast cells. Mammary fibroblast cells were prepared from FVB-Cdkn2a -/- ( Ink4a/Afr null) female mice as described in Materials and Methods, and superTOP-flash reporter assays were performed as above. Error bars = standard deviation of triplicate samples.
    Figure Legend Snippet: Wnt4 does not induce canonical Wnt signaling in mammary epithelial or fibroblast cells in vitro . (A) Wnt reporter assay of primary mammary epithelial cells. Primary mammary epithelial cells were transfected with superTOP-flash reporter gene, a Renilla luciferase gene (a transfection control), and various amounts of Wnt4 expression vector (0.05~2.0 μg). Luciferase activity was measured 48 hrs after transfection, and results corrected for transfection efficiency (dividing by Renilla luciferase activity). Fold induction was calculated with respect to the negative control ( LacZ -transfected cells). A Wnt1 expression vector was used as a positive control. (B) Wnt reporter assay of immortalized mammary fibroblast cells. Mammary fibroblast cells were prepared from FVB-Cdkn2a -/- ( Ink4a/Afr null) female mice as described in Materials and Methods, and superTOP-flash reporter assays were performed as above. Error bars = standard deviation of triplicate samples.

    Techniques Used: In Vitro, Reporter Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Negative Control, Positive Control, Mouse Assay, Standard Deviation

    15) Product Images from "Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus"

    Article Title: Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20012023

    Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.

    Techniques Used: Activation Assay, Mouse Assay, Infection, Isolation, Amplification, Polymerase Chain Reaction, Positive Control, Labeling, Injection

    Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).
    Figure Legend Snippet: Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).

    Techniques Used: Activation Assay, Mouse Assay, Labeling, Infection, Expressing, Staining

    Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.
    Figure Legend Snippet: Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.

    Techniques Used: In Vivo, CTL Assay, Activity Assay, Infection, Labeling, Mouse Assay, Isolation, Fluorescence, Concentration Assay, Lysis

    De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.

    Techniques Used: Mouse Assay, Labeling, Infection, Mutagenesis, Staining

    16) Product Images from "Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus"

    Article Title: Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20012023

    Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.

    Techniques Used: Activation Assay, Mouse Assay, Infection, Isolation, Amplification, Polymerase Chain Reaction, Positive Control, Labeling, Injection

    Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).
    Figure Legend Snippet: Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).

    Techniques Used: Activation Assay, Mouse Assay, Labeling, Infection, Expressing, Staining

    Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.
    Figure Legend Snippet: Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.

    Techniques Used: In Vivo, CTL Assay, Activity Assay, Infection, Labeling, Mouse Assay, Isolation, Fluorescence, Concentration Assay, Lysis

    De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.

    Techniques Used: Mouse Assay, Labeling, Infection, Mutagenesis, Staining

    17) Product Images from "EWI-2 regulates ?3?1 integrin-dependent cell functions on laminin-5"

    Article Title: EWI-2 regulates ?3?1 integrin-dependent cell functions on laminin-5

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200309113

    Tetraspanins support EWI-2- α 3 β 1 integrin association. (A) ∼10 7 A431 EWI-2 cells were labeled with sulfo-NHS biotin and lysed in 1% Brij 96. Equal portions of lysate were depleted three times with protein G alone (mock) or with protein G plus anti- tetraspanin or α2 integrin antibodies. Depletions are indicated at the top of the figure. Depleted lysates were further divided and immunoprecipitated with anti-CD81, anti-α3 integrin, or anti-α2 integrin antibodies conjugated to agarose followed by SDS-PAGE and blotting with HRP-ExtrAvidin ® . EWI-2* is a 50-kD EWI-2 cleavage fragment. Densitometry revealed that CD9 and CD81 depletion removed only ∼10% of total α3 integrin, but ∼85% of the α3-associated EWI-2. Densitometry of an independent experiment confirmed that only ∼10% of α3 is CD81-associated in A431 cells (not depicted). (B) U937 cells lacking CD9 and CD81 were transduced with CD81 and EWI-2 retroviral expression vectors and selected to obtain stable (CD81−, EWI-2+) or (CD81+, EWI-2+) cell lines. Then, both cell types were super-infected with an α3 integrin retroviral expression vector, to yield equivalent α3 expression levels, as confirmed by flow cytometry (not depicted). EWI-2 (M2 anti-FLAG mAb), CD81 (M38 mAb), or α3 integrin (A3X8 mAb) were immunoprecipitated from 1% Brij 96 lysates of CD81 − or CD81 + cells. Immunoprecipitates were blotted for EWI-2 (biotinylated M2 anti-FLAG mAb), α3 integrin (D23 pAb), or CD81 (M38 mAb). Apparent mature and immature forms of EWI-2 and α3 integrin are indicated with filled and open arrows, respectively. 6 × 10 6 cell equivalents were analyzed in EWI-2 and CD81 immunoprecipitations, and 4.8 × 10 7 cell equivalents in the α3 immunoprecipitations. (C) Biotinylated CD81 − or CD81 + cells were lysed in 1% Brij 96, and EWI-2 (FLAG), α3 integrin, or CD81 were immunoprecipitated as above. Cell surface–labeled proteins were revealed by HRP-ExtrAvidin ® blot. 1.7 × 10 6 cell equivalents were analyzed in each immunoprecipitation.
    Figure Legend Snippet: Tetraspanins support EWI-2- α 3 β 1 integrin association. (A) ∼10 7 A431 EWI-2 cells were labeled with sulfo-NHS biotin and lysed in 1% Brij 96. Equal portions of lysate were depleted three times with protein G alone (mock) or with protein G plus anti- tetraspanin or α2 integrin antibodies. Depletions are indicated at the top of the figure. Depleted lysates were further divided and immunoprecipitated with anti-CD81, anti-α3 integrin, or anti-α2 integrin antibodies conjugated to agarose followed by SDS-PAGE and blotting with HRP-ExtrAvidin ® . EWI-2* is a 50-kD EWI-2 cleavage fragment. Densitometry revealed that CD9 and CD81 depletion removed only ∼10% of total α3 integrin, but ∼85% of the α3-associated EWI-2. Densitometry of an independent experiment confirmed that only ∼10% of α3 is CD81-associated in A431 cells (not depicted). (B) U937 cells lacking CD9 and CD81 were transduced with CD81 and EWI-2 retroviral expression vectors and selected to obtain stable (CD81−, EWI-2+) or (CD81+, EWI-2+) cell lines. Then, both cell types were super-infected with an α3 integrin retroviral expression vector, to yield equivalent α3 expression levels, as confirmed by flow cytometry (not depicted). EWI-2 (M2 anti-FLAG mAb), CD81 (M38 mAb), or α3 integrin (A3X8 mAb) were immunoprecipitated from 1% Brij 96 lysates of CD81 − or CD81 + cells. Immunoprecipitates were blotted for EWI-2 (biotinylated M2 anti-FLAG mAb), α3 integrin (D23 pAb), or CD81 (M38 mAb). Apparent mature and immature forms of EWI-2 and α3 integrin are indicated with filled and open arrows, respectively. 6 × 10 6 cell equivalents were analyzed in EWI-2 and CD81 immunoprecipitations, and 4.8 × 10 7 cell equivalents in the α3 immunoprecipitations. (C) Biotinylated CD81 − or CD81 + cells were lysed in 1% Brij 96, and EWI-2 (FLAG), α3 integrin, or CD81 were immunoprecipitated as above. Cell surface–labeled proteins were revealed by HRP-ExtrAvidin ® blot. 1.7 × 10 6 cell equivalents were analyzed in each immunoprecipitation.

    Techniques Used: Labeling, Immunoprecipitation, SDS Page, Transduction, Expressing, Infection, Plasmid Preparation, Flow Cytometry, Cytometry

    Biochemical analysis of the EW2xCD2c chimera. (A) Wild-type and mutant EWI proteins were immunoprecipitated using anti-FLAG antibody, and then samples were analyzed by blotting using anti-α3, anti-CD81 (M38), and anti-FLAG pAbs. CD81 coprecipitating with EWI-2 is indicated with a small black arrow (middle). On the bottom, dark arrows indicate mature EWI proteins, and white arrows indicate immature forms. (B) From Brij 96 lysates of 10 6 cell surface–biotinylated, transduced A431 cells, EWI proteins were immunoprecipitated using anti-FLAG antibody, and then analyzed by HRP-ExtrAvidin ® blotting. The locations of α3 integrin, intact EW2xCD2c, intact EWI-2, CD9, and CD81 are indicated. Two exposures of the same blot are presented to allow comparison of the relative amounts of α3, CD9, and CD81 that coprecipitate with each protein. Note that intact EW2xCD2 (∼78 kD) and intact EWI-2 (∼70 kD) are each accompanied by two smaller cleavage products. (C) 3.6 × 10 6 A431 cells expressing EW2xCD2c or EWI-2were lysed in Brij 96, and the indicated proteins were immunoprecipitated. Samples were then immunoblotted for FLAG (M2 anti-FLAG mAb) or CD81 as above. In all cases, small differences in total protein in the lysates were measured by amido black assay and corrected before immunoprecipitation. Lanes 1* and 2* represent shorter exposures of lanes 5 and 6, respectively. (D) α3 integrin was immunoprecipitated from Brij 96 lysates of 3.3 × 10 5 transduced A431 cells. Levels of CD81 and α3 within the immunoprecipitates were revealed by immunoblotting with M38 anti-CD81 mAb (top) or with anti-α3 pAb (bottom).
    Figure Legend Snippet: Biochemical analysis of the EW2xCD2c chimera. (A) Wild-type and mutant EWI proteins were immunoprecipitated using anti-FLAG antibody, and then samples were analyzed by blotting using anti-α3, anti-CD81 (M38), and anti-FLAG pAbs. CD81 coprecipitating with EWI-2 is indicated with a small black arrow (middle). On the bottom, dark arrows indicate mature EWI proteins, and white arrows indicate immature forms. (B) From Brij 96 lysates of 10 6 cell surface–biotinylated, transduced A431 cells, EWI proteins were immunoprecipitated using anti-FLAG antibody, and then analyzed by HRP-ExtrAvidin ® blotting. The locations of α3 integrin, intact EW2xCD2c, intact EWI-2, CD9, and CD81 are indicated. Two exposures of the same blot are presented to allow comparison of the relative amounts of α3, CD9, and CD81 that coprecipitate with each protein. Note that intact EW2xCD2 (∼78 kD) and intact EWI-2 (∼70 kD) are each accompanied by two smaller cleavage products. (C) 3.6 × 10 6 A431 cells expressing EW2xCD2c or EWI-2were lysed in Brij 96, and the indicated proteins were immunoprecipitated. Samples were then immunoblotted for FLAG (M2 anti-FLAG mAb) or CD81 as above. In all cases, small differences in total protein in the lysates were measured by amido black assay and corrected before immunoprecipitation. Lanes 1* and 2* represent shorter exposures of lanes 5 and 6, respectively. (D) α3 integrin was immunoprecipitated from Brij 96 lysates of 3.3 × 10 5 transduced A431 cells. Levels of CD81 and α3 within the immunoprecipitates were revealed by immunoblotting with M38 anti-CD81 mAb (top) or with anti-α3 pAb (bottom).

    Techniques Used: Mutagenesis, Immunoprecipitation, Expressing

    Quantitation of A431 IZ cells and A431 EWI-2 cell spreading assays on laminin-5. The areas of 20–45 cells per coverslip were measured using Scion Image v1.62 software. Specific spreading was calculated by subtracting the mean area of cells fixed immediately after plating from the mean area of cells fixed at the end of the assay. Error bars indicate the SEM for each coverslip. Two coverslips per condition were examined; results for each coverslip are shown individually. Asterisk Indicates statistically significant difference between antibody-treated cells and the coverslips in the no antibody controls (P
    Figure Legend Snippet: Quantitation of A431 IZ cells and A431 EWI-2 cell spreading assays on laminin-5. The areas of 20–45 cells per coverslip were measured using Scion Image v1.62 software. Specific spreading was calculated by subtracting the mean area of cells fixed immediately after plating from the mean area of cells fixed at the end of the assay. Error bars indicate the SEM for each coverslip. Two coverslips per condition were examined; results for each coverslip are shown individually. Asterisk Indicates statistically significant difference between antibody-treated cells and the coverslips in the no antibody controls (P

    Techniques Used: Quantitation Assay, Software

    EWI-2 associates with tetraspanins and α 3 β 1 integrin. (A) A431 EWI-2 cells (∼5 × 10 6 ) expressing FLAG-tagged EWI-2 were lysed in 1% Brij 96. Equal volumes of the lysate were used for immunoprecipitations with anti-FLAG agarose or with anti-integrin or -tetraspanin antibodies. The blot was developed with biotinylated anti-FLAG antibody followed by HRP-ExtrAvidin ® to detect coprecipitating EWI-2. (B) A431 EWI-2 cells (EWI-2), A431 IZ cells (IZ), and A431 parental cells (P) were labeled with sulfo-NHS biotin and lysed in 1% Brij 96. Lysates were immunoprecipitated with anti-FLAG agarose or antibodies to the indicated tetraspanins and integrins, followed by blotting with HRP-ExtrAvidin ® . EWI-2* designates a 50-kD EWI-2 cleavage product, and two electrophoretic forms of CD9 are indicated. ∼7 × 10 5 cell equivalents were used for each immunoprecipitation. Flow cytometry results confirm that CD9, CD81, CD151, and α3β1 expression levels are similar in the different cell types. The bands of ∼210 kD in the α6 lanes (16–18) and CD151 lanes (10–12) correspond to integrin β4, a major α6 partner in A431 cells ( Rabinovitz et al., 1999 ). α6β4 is known to associate with CD151 ( Sterk et al., 2000 ). Densitometry indicated an approximate fourfold increase in α3-associated EWI-2 upon EWI-2 overexpression. Similar results were obtained in two independent experiments.
    Figure Legend Snippet: EWI-2 associates with tetraspanins and α 3 β 1 integrin. (A) A431 EWI-2 cells (∼5 × 10 6 ) expressing FLAG-tagged EWI-2 were lysed in 1% Brij 96. Equal volumes of the lysate were used for immunoprecipitations with anti-FLAG agarose or with anti-integrin or -tetraspanin antibodies. The blot was developed with biotinylated anti-FLAG antibody followed by HRP-ExtrAvidin ® to detect coprecipitating EWI-2. (B) A431 EWI-2 cells (EWI-2), A431 IZ cells (IZ), and A431 parental cells (P) were labeled with sulfo-NHS biotin and lysed in 1% Brij 96. Lysates were immunoprecipitated with anti-FLAG agarose or antibodies to the indicated tetraspanins and integrins, followed by blotting with HRP-ExtrAvidin ® . EWI-2* designates a 50-kD EWI-2 cleavage product, and two electrophoretic forms of CD9 are indicated. ∼7 × 10 5 cell equivalents were used for each immunoprecipitation. Flow cytometry results confirm that CD9, CD81, CD151, and α3β1 expression levels are similar in the different cell types. The bands of ∼210 kD in the α6 lanes (16–18) and CD151 lanes (10–12) correspond to integrin β4, a major α6 partner in A431 cells ( Rabinovitz et al., 1999 ). α6β4 is known to associate with CD151 ( Sterk et al., 2000 ). Densitometry indicated an approximate fourfold increase in α3-associated EWI-2 upon EWI-2 overexpression. Similar results were obtained in two independent experiments.

    Techniques Used: Expressing, Labeling, Immunoprecipitation, Flow Cytometry, Cytometry, Over Expression

    Impaired migration of A431 EWI-2 cells on laminin-5. (A) In two separate trials, XY locations of individual cells were determined once every 10 min using Scion Image v1.62 software, and the average migration rate was calculated from the series of XY measurements using KaleidaGraph v3.05 software (Abelbeck Software). A431 EWI-2 cells migration rates were 60–70% lower than those of A431 IZ control cells (*, P
    Figure Legend Snippet: Impaired migration of A431 EWI-2 cells on laminin-5. (A) In two separate trials, XY locations of individual cells were determined once every 10 min using Scion Image v1.62 software, and the average migration rate was calculated from the series of XY measurements using KaleidaGraph v3.05 software (Abelbeck Software). A431 EWI-2 cells migration rates were 60–70% lower than those of A431 IZ control cells (*, P

    Techniques Used: Migration, Software

    EWI-2 expression impairs A431 cell reaggregation on laminin-5. A431 IZ control or EWI-2 cells were plated, after enzymatic dissociation, in serum-free medium on coverslips coated with laminin-5 (A–C) or collagen I (D–F). After 5 h, cells were fixed and photographed in phase contrast, revealing several clusters of four or more A431 IZ cells in most fields, as indicated by asterisks in A. By contrast, few clusters of A431 EWI-2 cells were observed in B. (C) Quantitation of three separate experiments (two coverslips per condition in each experiment, five fields per coverslip) revealed a statistically significant decrease in the percentage of clustered A431 EWI-2 cells on laminin-5 (P
    Figure Legend Snippet: EWI-2 expression impairs A431 cell reaggregation on laminin-5. A431 IZ control or EWI-2 cells were plated, after enzymatic dissociation, in serum-free medium on coverslips coated with laminin-5 (A–C) or collagen I (D–F). After 5 h, cells were fixed and photographed in phase contrast, revealing several clusters of four or more A431 IZ cells in most fields, as indicated by asterisks in A. By contrast, few clusters of A431 EWI-2 cells were observed in B. (C) Quantitation of three separate experiments (two coverslips per condition in each experiment, five fields per coverslip) revealed a statistically significant decrease in the percentage of clustered A431 EWI-2 cells on laminin-5 (P

    Techniques Used: Expressing, Quantitation Assay

    EWI-2 overexpression does not affect A431 cell spreading on laminin-5. A431 IZ control cells or A431 EWI-2 cells were plated in serum-free medium on laminin-5–coated coverslips with no antibodies (A and B), or in the presence of 5 μg/ml GoH3 anti-α6 integrin (C and D), 10 μg/ml A3-IIF5 anti-α3 integrin (E and F), or both anti-α3 and -α6 antibodies (G and H). Cells were fixed after 20 min and photographed with phase contrast. Inset in B shows anti-FLAG EWI-2 immunoprecipitate from cell surface–biotinylated A431 EWI-2 cells (EWI-2) or A431 IZ control cells (IZ) developed with HRP-ExtrAvidin ® . Mol wt markers (kD) are indicated.
    Figure Legend Snippet: EWI-2 overexpression does not affect A431 cell spreading on laminin-5. A431 IZ control cells or A431 EWI-2 cells were plated in serum-free medium on laminin-5–coated coverslips with no antibodies (A and B), or in the presence of 5 μg/ml GoH3 anti-α6 integrin (C and D), 10 μg/ml A3-IIF5 anti-α3 integrin (E and F), or both anti-α3 and -α6 antibodies (G and H). Cells were fixed after 20 min and photographed with phase contrast. Inset in B shows anti-FLAG EWI-2 immunoprecipitate from cell surface–biotinylated A431 EWI-2 cells (EWI-2) or A431 IZ control cells (IZ) developed with HRP-ExtrAvidin ® . Mol wt markers (kD) are indicated.

    Techniques Used: Over Expression

    Altered CD81 localization in A431 EWI-2 cells. A431 IZ, A431 EW2xCD2c, and A431 EWI-2 cells were plated on laminin-5–coated coverslips in serum-free medium and fixed after 3.5 h. Cells were stained for α3 integrin with the A3X8 mAb (A–C), CD81 with the JS64 mAb (D–F and J–L), or α4 integrin with the B5G10 mAb (M). Staining was visualized with FITC-goat anti–mouse secondary antibody. Although α3 localization appeared similar in all three cell types, CD81 was strongly concentrated in filopodia at the perimeters of A431 EWI-2 cells. G–I shows phase-contrast images of the same fields as in D–F, to reveal cell morphology. J–L show expansion of regions boxed in D–F and G–I. Arrows in D, E, G, and H indicate a subset of cells with a round, symmetrical morphology that nevertheless failed to demonstrate the striking concentration of CD9 and CD81 at the cell perimeter that was observed in the EWI-2–overexpressing cells.
    Figure Legend Snippet: Altered CD81 localization in A431 EWI-2 cells. A431 IZ, A431 EW2xCD2c, and A431 EWI-2 cells were plated on laminin-5–coated coverslips in serum-free medium and fixed after 3.5 h. Cells were stained for α3 integrin with the A3X8 mAb (A–C), CD81 with the JS64 mAb (D–F and J–L), or α4 integrin with the B5G10 mAb (M). Staining was visualized with FITC-goat anti–mouse secondary antibody. Although α3 localization appeared similar in all three cell types, CD81 was strongly concentrated in filopodia at the perimeters of A431 EWI-2 cells. G–I shows phase-contrast images of the same fields as in D–F, to reveal cell morphology. J–L show expansion of regions boxed in D–F and G–I. Arrows in D, E, G, and H indicate a subset of cells with a round, symmetrical morphology that nevertheless failed to demonstrate the striking concentration of CD9 and CD81 at the cell perimeter that was observed in the EWI-2–overexpressing cells.

    Techniques Used: Staining, Concentration Assay

    18) Product Images from "Cervical Lymph Nodes as a Selective Niche for Brucella during Oral Infections"

    Article Title: Cervical Lymph Nodes as a Selective Niche for Brucella during Oral Infections

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121790

    B . melitensis oral infection results in an increase of CLN CD11b high cells expressing either F4/80, Ly6c or both. CLN from mice orally infected with 10 9 B . melitensis per mouse for 15 days were prepared for flow cytometry. Total CLN cell numbers were analyzed for the respective percentages (A) or absolute numbers (B) of dendritic cells (F4/80 - CD11c high MHCII int and F4/80 - CD11c int MHCII high ), CD11b high macrophages/monocytes (F4/80 - Ly6c + , F4/80 + Ly6c + and F4/80 + Ly6c - ) and neutrophils (CD11b high Ly6G + ). (C) shows a representative contour plot of respective populations from a mock-infected or Brucella -infected mouse on CD19 - Ly6G - CD11b high (F4/80 vs. Ly6c) or CD19 - CD11b low/int F4/80 - NK1.1 - cells (CD11c vs. MHCII). Populations shown in (A) were analyzed for their median fluorescence of (D) CD11b, (E) CD11c and (F) MHCII. Data represent mean and SEM of pooled results from two independent experiments with a total of 8 (mock-infected) and 9 (infected) mice per group. * p ≤ 0.05 as compared to respective mock infected control.
    Figure Legend Snippet: B . melitensis oral infection results in an increase of CLN CD11b high cells expressing either F4/80, Ly6c or both. CLN from mice orally infected with 10 9 B . melitensis per mouse for 15 days were prepared for flow cytometry. Total CLN cell numbers were analyzed for the respective percentages (A) or absolute numbers (B) of dendritic cells (F4/80 - CD11c high MHCII int and F4/80 - CD11c int MHCII high ), CD11b high macrophages/monocytes (F4/80 - Ly6c + , F4/80 + Ly6c + and F4/80 + Ly6c - ) and neutrophils (CD11b high Ly6G + ). (C) shows a representative contour plot of respective populations from a mock-infected or Brucella -infected mouse on CD19 - Ly6G - CD11b high (F4/80 vs. Ly6c) or CD19 - CD11b low/int F4/80 - NK1.1 - cells (CD11c vs. MHCII). Populations shown in (A) were analyzed for their median fluorescence of (D) CD11b, (E) CD11c and (F) MHCII. Data represent mean and SEM of pooled results from two independent experiments with a total of 8 (mock-infected) and 9 (infected) mice per group. * p ≤ 0.05 as compared to respective mock infected control.

    Techniques Used: Infection, Expressing, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence

    19) Product Images from "Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts"

    Article Title: Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033626

    Activation profiles of mTOR signaling in the interstitial inflammatory cells. Kidney sections derived from either UUO or IRI models were analyzed by immunohistochemistry using antibodies against pS6K, F4/80, CD4 or anti-neutrophil. A–A′″ and B–B′″: Expression of pS6K (red) is immediately induced in F4/80+ macrophages (green) after kidney injury. Large portions of interstitial macrophages in obstructed kidney (A–A′″) and IRI kidney (B–B″) are co-stained with F4/80 and pS6K (indicated by arrwos). C–E: Co-staining of CD4 (green) and pS6K in obstructed kidneys. F: Costaining of anti-neutrophil and pS6K in obstructed kidneys. Scale bar: 20 µm.
    Figure Legend Snippet: Activation profiles of mTOR signaling in the interstitial inflammatory cells. Kidney sections derived from either UUO or IRI models were analyzed by immunohistochemistry using antibodies against pS6K, F4/80, CD4 or anti-neutrophil. A–A′″ and B–B′″: Expression of pS6K (red) is immediately induced in F4/80+ macrophages (green) after kidney injury. Large portions of interstitial macrophages in obstructed kidney (A–A′″) and IRI kidney (B–B″) are co-stained with F4/80 and pS6K (indicated by arrwos). C–E: Co-staining of CD4 (green) and pS6K in obstructed kidneys. F: Costaining of anti-neutrophil and pS6K in obstructed kidneys. Scale bar: 20 µm.

    Techniques Used: Activation Assay, Derivative Assay, Immunohistochemistry, Expressing, Staining

    Rapamycin inhibits inflammatory activity of macrophages isolated from obstructed kidneys. A–B: Co-immunostainging images of kidney sections from day-1 post-obstruction with administration of rapamycin (A–A′″) or vehicle (B–B′″), using anti-pS6K (red), anti-F4/80 (green) and DAPI (blue) for immunofluorescent staining. Representative areas in A″ and B″ (indicated by white square) are magnified in A′″ and B′″, respectively. Scale bar: 50 µm. C. Assessment of inflammatory activity of isolated macrophages from obstructed kidney on day-1 post-operation. mRNA level of proinflammatory chemokines, including TNF-α, IL-1β and MCP-1, were determined by realtime-PCR. *P
    Figure Legend Snippet: Rapamycin inhibits inflammatory activity of macrophages isolated from obstructed kidneys. A–B: Co-immunostainging images of kidney sections from day-1 post-obstruction with administration of rapamycin (A–A′″) or vehicle (B–B′″), using anti-pS6K (red), anti-F4/80 (green) and DAPI (blue) for immunofluorescent staining. Representative areas in A″ and B″ (indicated by white square) are magnified in A′″ and B′″, respectively. Scale bar: 50 µm. C. Assessment of inflammatory activity of isolated macrophages from obstructed kidney on day-1 post-operation. mRNA level of proinflammatory chemokines, including TNF-α, IL-1β and MCP-1, were determined by realtime-PCR. *P

    Techniques Used: Activity Assay, Isolation, Staining, Polymerase Chain Reaction

    Rapamycin attenuates inflammatory responses in obstructed kidneys. A–B: Immunoflurescent staining (A) and quantitative assessment (B) of F4/80+ macrophages in kidney sections from UUO mouse models. anti-F4/80 (green) was used to label macrophages in kidney tissues, costaining with DAPI (blue). Scale bar: 20 µm. ** P
    Figure Legend Snippet: Rapamycin attenuates inflammatory responses in obstructed kidneys. A–B: Immunoflurescent staining (A) and quantitative assessment (B) of F4/80+ macrophages in kidney sections from UUO mouse models. anti-F4/80 (green) was used to label macrophages in kidney tissues, costaining with DAPI (blue). Scale bar: 20 µm. ** P

    Techniques Used: Staining

    20) Product Images from "Adenovirus Type 5 Induces Vitamin A-Metabolizing Enzymes in Dendritic Cells and Enhances Priming of Gut Homing CD8 T Cells"

    Article Title: Adenovirus Type 5 Induces Vitamin A-Metabolizing Enzymes in Dendritic Cells and Enhances Priming of Gut Homing CD8 T Cells

    Journal: Mucosal immunology

    doi: 10.1038/mi.2011.1

    Ad5 endows splenic conventional DC (cDC) with the capacity to imprint gut homing potential on antigen-specific CD8 T cells via a retinoic acid (RA) dependent pathway Splenic cDCs isolated from C57BL/6 mice were either left uninfected or infected with Ad5 (non-recombinant) for 8hrs. Following infection, cells were washed and co-cultured with OT-I T cells (CD8) in the presence of the OVA peptide for 72 hrs. (A) Representative FACS plots (left panel) showing cell surface expression of α4β7on OT-I T cells 3 days after co-culture. Cells were gated on total CD8 T cells. Middle panel, mean frequency of α4β7 + cells (gated on total live CD8 lymphocytes) following co-culture with either uninfected cDCs or Ad5-infected cDCs at indicated MOIs. Right Panel, total live (Via-Probe negative) CD8 T cell number at the end of the co-culture primed by cDCs infected with different MOIs. (B) Mean frequency of CCR9 + cells (gated on total live CD8 lymphocytes) following co-culture with either uninfected cDCs or Ad5-infected cDCs at MOI 2. (C) Representative FACS plots (left panel) showing α4β7 expression on total live CD8 T cells following co-culture with Ad5-infected cDCs (MOI 0.15) in the absence or presence of retinoic acid receptor (RAR) inhibitor LE540. Middle panel, frequency of α4β7 + CD8 T cells in the absence or presence of LE540 (inhibitor included in the co-culture). Total live (Via-Probe negative) CD8 T cell number in the absence or presence of LE540 at the end of the co-culture. Data from two independent experiments (Exp.1 and Exp.2) are shown. Right panel, frequency ofα4β7 + CD8 T cells with RAR inhibitor included either only during 8hrs of Ad5 infection and washed before co-culture (pre-treat) or left in the well during co-culture for 72hrs (in well). (D) Quantitative RT-PCR showing the fold change (mean of six independent experiments) in the expression of mRNA encoding retinal dehydrogenase (RALDH) enzymes ( Aldh1a1 , Aldh1a2 and Aldh1a3 ) in Ad5-infected cDCs over uninfected cDCs at 0, 6–12 and 24 hrs following in vitro infection. (E) Representative FACS plots (left panel) showing ALDH+ cells (gated on total CD11c+ cells) at 8hrs following in vitro Ad5 infection. The ALDH+ gate was determined based on the DEAB treated uninfected sample. Right panel, cumulative data (mean) for three independent experiments. (F) Mean frequency of α4β7 + CD8 T cells following co-culture with either uninfected cDCs or Ad5-infected cDCs in the absence or presence of a RALDH inhibitor, DEAB (left panel). Right panel, total live (Via-Probe negative) CD8 T cell number in the absence or presence of DEAB at the end of the co-culture. (G) C57BL/6 mice were immunized with Ad5 (5X10 7 pfu per mouse) intramuscularly and draining lymph nodes (inguinal and popliteal pooled) were harvested at 12hrs post immunization. Quantitative RT-PCR showing the fold change in the expression of mRNA encoding retinal dehydrogenase (RALDH) enzymes ( Aldh1a1 , Aldh1a2 and Aldh1a3 ) in cDCs from Ad5-infected mice DLN over naïve mice DLN. Results from two independent experiments are shown (Exp.1 and Exp.2). Error bars indicate SEM. All data (except Fig. 2G) are representative of at least three independent experiments. * P
    Figure Legend Snippet: Ad5 endows splenic conventional DC (cDC) with the capacity to imprint gut homing potential on antigen-specific CD8 T cells via a retinoic acid (RA) dependent pathway Splenic cDCs isolated from C57BL/6 mice were either left uninfected or infected with Ad5 (non-recombinant) for 8hrs. Following infection, cells were washed and co-cultured with OT-I T cells (CD8) in the presence of the OVA peptide for 72 hrs. (A) Representative FACS plots (left panel) showing cell surface expression of α4β7on OT-I T cells 3 days after co-culture. Cells were gated on total CD8 T cells. Middle panel, mean frequency of α4β7 + cells (gated on total live CD8 lymphocytes) following co-culture with either uninfected cDCs or Ad5-infected cDCs at indicated MOIs. Right Panel, total live (Via-Probe negative) CD8 T cell number at the end of the co-culture primed by cDCs infected with different MOIs. (B) Mean frequency of CCR9 + cells (gated on total live CD8 lymphocytes) following co-culture with either uninfected cDCs or Ad5-infected cDCs at MOI 2. (C) Representative FACS plots (left panel) showing α4β7 expression on total live CD8 T cells following co-culture with Ad5-infected cDCs (MOI 0.15) in the absence or presence of retinoic acid receptor (RAR) inhibitor LE540. Middle panel, frequency of α4β7 + CD8 T cells in the absence or presence of LE540 (inhibitor included in the co-culture). Total live (Via-Probe negative) CD8 T cell number in the absence or presence of LE540 at the end of the co-culture. Data from two independent experiments (Exp.1 and Exp.2) are shown. Right panel, frequency ofα4β7 + CD8 T cells with RAR inhibitor included either only during 8hrs of Ad5 infection and washed before co-culture (pre-treat) or left in the well during co-culture for 72hrs (in well). (D) Quantitative RT-PCR showing the fold change (mean of six independent experiments) in the expression of mRNA encoding retinal dehydrogenase (RALDH) enzymes ( Aldh1a1 , Aldh1a2 and Aldh1a3 ) in Ad5-infected cDCs over uninfected cDCs at 0, 6–12 and 24 hrs following in vitro infection. (E) Representative FACS plots (left panel) showing ALDH+ cells (gated on total CD11c+ cells) at 8hrs following in vitro Ad5 infection. The ALDH+ gate was determined based on the DEAB treated uninfected sample. Right panel, cumulative data (mean) for three independent experiments. (F) Mean frequency of α4β7 + CD8 T cells following co-culture with either uninfected cDCs or Ad5-infected cDCs in the absence or presence of a RALDH inhibitor, DEAB (left panel). Right panel, total live (Via-Probe negative) CD8 T cell number in the absence or presence of DEAB at the end of the co-culture. (G) C57BL/6 mice were immunized with Ad5 (5X10 7 pfu per mouse) intramuscularly and draining lymph nodes (inguinal and popliteal pooled) were harvested at 12hrs post immunization. Quantitative RT-PCR showing the fold change in the expression of mRNA encoding retinal dehydrogenase (RALDH) enzymes ( Aldh1a1 , Aldh1a2 and Aldh1a3 ) in cDCs from Ad5-infected mice DLN over naïve mice DLN. Results from two independent experiments are shown (Exp.1 and Exp.2). Error bars indicate SEM. All data (except Fig. 2G) are representative of at least three independent experiments. * P

    Techniques Used: Isolation, Mouse Assay, Infection, Recombinant, Cell Culture, FACS, Expressing, Co-Culture Assay, Quantitative RT-PCR, In Vitro

    21) Product Images from "DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers"

    Article Title: DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200910191

    Representative confocal x-y images (1/second) obtained from fluo-4–loaded FDB fibers before, during, and after exposure to solutions of increased or decreased osmolarity. For each cell, the number of LCR events observed per frame throughout the experiment is indicated in the graph on the right. (A) The application of a hyperosmotic sucrose solution was often associated with a marked elevation of [Ca 2+ ], and LCR was difficult to identify (cell 1). However, where the rise in [Ca 2+ ] was less pronounced, individual LCR events were apparent during the application of sucrose (cell 2). Prior exposure to 100 µM nifedipine, to inhibit the DHPR, prevented LCR upon sucrose exposure (cell 3). (B) The introduction of a hyperosmotic CaCl 2 solution induced LCR (cell 1), which was markedly inhibited in the presence of nifedipine (cell 2). (C) The application of a hypoosmotic (254 mOsm) solution had no apparent effect on [Ca 2+ ] i . However, returning to the isoosmotic solution precipitated LCR events (cell 1). In the presence of nifedipine, transient exposure to a hypoosmotic solution failed to induce LCR (cell 2). Fibers subject to hypoosmotic Tyrode's exposure were initially exposed to isoosmotic Tyrode for 30 s, and then for 2 min to a hypoosmotic before returning to isoosmotic Tyrode's solution for 4 min. Horizontal bar, 20 µm.
    Figure Legend Snippet: Representative confocal x-y images (1/second) obtained from fluo-4–loaded FDB fibers before, during, and after exposure to solutions of increased or decreased osmolarity. For each cell, the number of LCR events observed per frame throughout the experiment is indicated in the graph on the right. (A) The application of a hyperosmotic sucrose solution was often associated with a marked elevation of [Ca 2+ ], and LCR was difficult to identify (cell 1). However, where the rise in [Ca 2+ ] was less pronounced, individual LCR events were apparent during the application of sucrose (cell 2). Prior exposure to 100 µM nifedipine, to inhibit the DHPR, prevented LCR upon sucrose exposure (cell 3). (B) The introduction of a hyperosmotic CaCl 2 solution induced LCR (cell 1), which was markedly inhibited in the presence of nifedipine (cell 2). (C) The application of a hypoosmotic (254 mOsm) solution had no apparent effect on [Ca 2+ ] i . However, returning to the isoosmotic solution precipitated LCR events (cell 1). In the presence of nifedipine, transient exposure to a hypoosmotic solution failed to induce LCR (cell 2). Fibers subject to hypoosmotic Tyrode's exposure were initially exposed to isoosmotic Tyrode for 30 s, and then for 2 min to a hypoosmotic before returning to isoosmotic Tyrode's solution for 4 min. Horizontal bar, 20 µm.

    Techniques Used:

    22) Product Images from "Rgs6 is Required for Adult Maintenance of Dopaminergic Neurons in the Ventral Substantia Nigra"

    Article Title: Rgs6 is Required for Adult Maintenance of Dopaminergic Neurons in the Ventral Substantia Nigra

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004863

    Subset-specific expression signatures of mDA neurons. (A) Differentially expressed probesets were clustered in an unbiased manner into three-dimensional Cartesian-type coordinates defined according to the relative enrichment attributes for each probeset in the comparisons between SNc WT/VTA WT, SNc KO/SNc WT and VTA KO/VTA WT. Each cluster was named according to the observed preferential expression pattern (label and shown in red in diagrams). The heatmaps show relative enrichments for 20 genes that are highly enriched and/or that have documented functions (the complete gene lists for each subset is provided in Table S1 ). The total number of genes (not probesets) in each cluster is indicated together with the number of those that are either activated or repressed by Pitx3 based on the VTA KO/WT comparison. (B) qRT-PCR quantification of mRNAs with preferential expression in either SN or VTA assessed in FACS-sorted cells from WT or Pitx3 −/− (KO) tissues. mRNA levels are normalized relative to Gapdh mRNA. Data are shown as means ± S.D.
    Figure Legend Snippet: Subset-specific expression signatures of mDA neurons. (A) Differentially expressed probesets were clustered in an unbiased manner into three-dimensional Cartesian-type coordinates defined according to the relative enrichment attributes for each probeset in the comparisons between SNc WT/VTA WT, SNc KO/SNc WT and VTA KO/VTA WT. Each cluster was named according to the observed preferential expression pattern (label and shown in red in diagrams). The heatmaps show relative enrichments for 20 genes that are highly enriched and/or that have documented functions (the complete gene lists for each subset is provided in Table S1 ). The total number of genes (not probesets) in each cluster is indicated together with the number of those that are either activated or repressed by Pitx3 based on the VTA KO/WT comparison. (B) qRT-PCR quantification of mRNAs with preferential expression in either SN or VTA assessed in FACS-sorted cells from WT or Pitx3 −/− (KO) tissues. mRNA levels are normalized relative to Gapdh mRNA. Data are shown as means ± S.D.

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, FACS

    Restricted expression of Rgs6 in Pitx3-positive (Pitx3+) dopaminergic neurons of ventral SNc. (A) Rgs6 expression revealed by immunohistofluorescence (red) together with TH (green) in mDA neurons of SNc but not VTA. Scale bar 410 µm. (B) Co-immunofluorescence analysis of TH (green) and Rgs6 or Pitx3 (red, as indicated) in SNc and VTA of adult mice. Arrowheads point to Pitx3-negative or Rgs6-negative mDA neurons in dorsal SNc. Scale bar 100 µm. (C) Triple immunofluorescence staining for TH (green), Pitx3 (red) and Calb1 (blue) on tissue sections of adult mice indicates that the majority of TH+Pitx3− cells of dSNc (arrowheads) are positive for Calb1, while TH+Pitx3+ cells of vSNc are negative for Calb1, consistent with depiction in A. Scale bar 100 µm.
    Figure Legend Snippet: Restricted expression of Rgs6 in Pitx3-positive (Pitx3+) dopaminergic neurons of ventral SNc. (A) Rgs6 expression revealed by immunohistofluorescence (red) together with TH (green) in mDA neurons of SNc but not VTA. Scale bar 410 µm. (B) Co-immunofluorescence analysis of TH (green) and Rgs6 or Pitx3 (red, as indicated) in SNc and VTA of adult mice. Arrowheads point to Pitx3-negative or Rgs6-negative mDA neurons in dorsal SNc. Scale bar 100 µm. (C) Triple immunofluorescence staining for TH (green), Pitx3 (red) and Calb1 (blue) on tissue sections of adult mice indicates that the majority of TH+Pitx3− cells of dSNc (arrowheads) are positive for Calb1, while TH+Pitx3+ cells of vSNc are negative for Calb1, consistent with depiction in A. Scale bar 100 µm.

    Techniques Used: Expressing, Immunohistofluorescence, Multiple Displacement Amplification, Immunofluorescence, Mouse Assay, Staining

    Unilateral loss of Pitx3-positive dopaminergic neurons in ventral SNc of Rgs6 −/− mice. (A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing less SNc TH+ neurons on one side of Rgs6 −/− mice at 1 year of age compared to sib control. Sections are identified with Bregma position. Scale bar 400 µm. (B) Number of TH+ cells in SNc and VTA of TH-stained coronal sections across midbrain (every 30 µm). Cell counts are represented as means +/− S.D. (***p
    Figure Legend Snippet: Unilateral loss of Pitx3-positive dopaminergic neurons in ventral SNc of Rgs6 −/− mice. (A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing less SNc TH+ neurons on one side of Rgs6 −/− mice at 1 year of age compared to sib control. Sections are identified with Bregma position. Scale bar 400 µm. (B) Number of TH+ cells in SNc and VTA of TH-stained coronal sections across midbrain (every 30 µm). Cell counts are represented as means +/− S.D. (***p

    Techniques Used: Mouse Assay, Immunoperoxidase Staining, Staining

    Strategy for isolation of FACS-purified Pitx3-dependent (red) and Pitx3-independent (white) mDA neurons for expression profiling analysis. Dissected SN and VTA from newborn TH-EGFP transgenic Pitx3 +/+ and Pitx3 −/ − mice were used for FACS purification of catecholaminergic neurons. The EGFP + cells consists in various proportions (approximate % shown) of Pitx3+ (red), Pitx3− (white) and Pitx3del (yellow) mDA neurons, depending on the region dissected and mDA neuronal loss resulting from Pitx3 inactivation. RNA from four cell preparations (SNc WT, VTA WT, SNc KO, VTA KO) were analyzed by hybridization in duplicates to Affymetrix Mouse Gene 1.0ST microarrays.
    Figure Legend Snippet: Strategy for isolation of FACS-purified Pitx3-dependent (red) and Pitx3-independent (white) mDA neurons for expression profiling analysis. Dissected SN and VTA from newborn TH-EGFP transgenic Pitx3 +/+ and Pitx3 −/ − mice were used for FACS purification of catecholaminergic neurons. The EGFP + cells consists in various proportions (approximate % shown) of Pitx3+ (red), Pitx3− (white) and Pitx3del (yellow) mDA neurons, depending on the region dissected and mDA neuronal loss resulting from Pitx3 inactivation. RNA from four cell preparations (SNc WT, VTA WT, SNc KO, VTA KO) were analyzed by hybridization in duplicates to Affymetrix Mouse Gene 1.0ST microarrays.

    Techniques Used: Isolation, FACS, Purification, Multiple Displacement Amplification, Expressing, Transgenic Assay, Mouse Assay, Hybridization

    Unilateral degeneration of vSNc neurons in a subset of Rgs6−/− mice. (A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing dysmorphic TH+ neurons (low TH staining, TH low , inset) in ventral SNc of Rgs6 −/− mice. The dSNc and VTA mDA neurons are unaffected (strong/normal TH staining, inset). Scale bar 200 µm. (B) Triple staining for TH (red), Dapi (blue) and Fluoro-Jade C (FJC, green) showing presence of degenerating TH low cells in vSNc of 1 y-old Rgs6 −/− mice (middle panels) and not in control vSNc (upper panels) or in dorsal SNc (lower panels). Scale bar 20 µm. (C) Bilateral cell counts of FJC + mDA neurons in SNc and VTA of coronal sections from control and 1 y-old Rgs6 −/− mice. (D) Co-immunostaining for TH (green) and LC3B (red) in vSNc of WT and Rgs6 −/− mice. Scale bar 20 µm. Arrowheads indicate unaffected neurons while arrows point to TH low cells. (E) Co-immunostaining for TH (green) and phosphorylated p27 Kip1 (phospho-p27, red) in vSNc of WT and Rgs6 −/− mice. Scale bar 20 µm.
    Figure Legend Snippet: Unilateral degeneration of vSNc neurons in a subset of Rgs6−/− mice. (A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing dysmorphic TH+ neurons (low TH staining, TH low , inset) in ventral SNc of Rgs6 −/− mice. The dSNc and VTA mDA neurons are unaffected (strong/normal TH staining, inset). Scale bar 200 µm. (B) Triple staining for TH (red), Dapi (blue) and Fluoro-Jade C (FJC, green) showing presence of degenerating TH low cells in vSNc of 1 y-old Rgs6 −/− mice (middle panels) and not in control vSNc (upper panels) or in dorsal SNc (lower panels). Scale bar 20 µm. (C) Bilateral cell counts of FJC + mDA neurons in SNc and VTA of coronal sections from control and 1 y-old Rgs6 −/− mice. (D) Co-immunostaining for TH (green) and LC3B (red) in vSNc of WT and Rgs6 −/− mice. Scale bar 20 µm. Arrowheads indicate unaffected neurons while arrows point to TH low cells. (E) Co-immunostaining for TH (green) and phosphorylated p27 Kip1 (phospho-p27, red) in vSNc of WT and Rgs6 −/− mice. Scale bar 20 µm.

    Techniques Used: Mouse Assay, Immunoperoxidase Staining, Staining, Multiple Displacement Amplification, Immunostaining

    Reduced expression of Pitx3 and its target genes in degenerating neurons. (A) Double immunofluorescence staining against TH (green) and Pitx3 (red) in SNc of control and 1 y-old Rgs6 −/− mice that display dysmorphic mDA neurons. Arrowheads indicate Pitx3-negative neurons of dSNc. Scale bar 20 µm. (B) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Vmat2 (red) in SNc of WT and Rgs6 −/− mice. Scale bar 20 µm. (C) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Bdnf (red) in SNc and VTA of WT and Rgs6 −/− mice. Scale bar 20 µm. (D) Double immunofluorescence staining against TH (green) and Pitx3, Aldh1a1, Fgf10 (red) in control and 1 y-old Rgs6 −/− mice that display dysmorphic mDA neurons. Arrowheads indicate unaffected dSNc neurons and arrows point to affected vSNc neurons. Scale bar 100 µm.
    Figure Legend Snippet: Reduced expression of Pitx3 and its target genes in degenerating neurons. (A) Double immunofluorescence staining against TH (green) and Pitx3 (red) in SNc of control and 1 y-old Rgs6 −/− mice that display dysmorphic mDA neurons. Arrowheads indicate Pitx3-negative neurons of dSNc. Scale bar 20 µm. (B) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Vmat2 (red) in SNc of WT and Rgs6 −/− mice. Scale bar 20 µm. (C) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Bdnf (red) in SNc and VTA of WT and Rgs6 −/− mice. Scale bar 20 µm. (D) Double immunofluorescence staining against TH (green) and Pitx3, Aldh1a1, Fgf10 (red) in control and 1 y-old Rgs6 −/− mice that display dysmorphic mDA neurons. Arrowheads indicate unaffected dSNc neurons and arrows point to affected vSNc neurons. Scale bar 100 µm.

    Techniques Used: Expressing, Double Immunofluorescence Staining, Mouse Assay, Multiple Displacement Amplification, Immunofluorescence, Staining

    23) Product Images from "Drebrin contains a cryptic F-actin-bundling activity regulated by Cdk5 phosphorylation"

    Article Title: Drebrin contains a cryptic F-actin-bundling activity regulated by Cdk5 phosphorylation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201303005

    Phosphorylation of drebrin at S142 is necessary and sufficient to relieve the intramolecular repression of F-actin bundling. (A) Immunofluorescence confocal images of COS-7 cells transfected with cDNA encoding YFP-tagged drebrin (green) mutated at S142 changing S to A (S142A) or S to D (S142D). Cells were labeled with phalloidin for F-actin (red). S142A drebrin-YFP only weakly induces F-actin containing filopodia, whereas S142D drebrin-YFP strongly induces filopodia (arrows). (B) Quantification of the number of filopodia per unit length of cell perimeter (filopodia density) in COS-7 cells transfected with cDNA encoding YFP, YFP-tagged drebrin (WT), and YFP-tagged drebrin mutated at S142 changing S to A (S142A) or S to D (S142D). Values are mean ± SEM (error bars) of 10 or more cells per transfection from three independent experiments. Significant differences (unpaired Student’s t test): *, P
    Figure Legend Snippet: Phosphorylation of drebrin at S142 is necessary and sufficient to relieve the intramolecular repression of F-actin bundling. (A) Immunofluorescence confocal images of COS-7 cells transfected with cDNA encoding YFP-tagged drebrin (green) mutated at S142 changing S to A (S142A) or S to D (S142D). Cells were labeled with phalloidin for F-actin (red). S142A drebrin-YFP only weakly induces F-actin containing filopodia, whereas S142D drebrin-YFP strongly induces filopodia (arrows). (B) Quantification of the number of filopodia per unit length of cell perimeter (filopodia density) in COS-7 cells transfected with cDNA encoding YFP, YFP-tagged drebrin (WT), and YFP-tagged drebrin mutated at S142 changing S to A (S142A) or S to D (S142D). Values are mean ± SEM (error bars) of 10 or more cells per transfection from three independent experiments. Significant differences (unpaired Student’s t test): *, P

    Techniques Used: Immunofluorescence, Transfection, Labeling

    Drebrin contains two F-actin–binding domains responsible for filopodia formation in heterologous cells. (A) Domain diagrams of drebrin and drebrin deletion constructs used for the functional domain analysis of drebrin. Drebrin has five potential functional domains identified by an in silico analysis using InterPro, Pfam, SMART, and PROSITE. Constructs are YFP-tagged at the C terminus. ADFH, actin-depolymerizing factor homology domain (red, residues 1–135); CC, coiled-coil domain (turquoise, residues 176–256); Hel, helical domain (orange, residues 256–355); PP, proline-rich region (green, residues 364–417); BB, blue box, drebrin C-terminal region (blue, residues 431–649). Numbering is for human drebrin E. (B–E) Immunofluorescence confocal images of COS-7 cells transfected with cDNA encoding YFP-tagged drebrin and drebrin deletion constructs (green) and labeled with phalloidin for F-actin (red). Bars, 5 µm. (B) COS-7 cells expressing YFP alone are rounded and have few processes. The YFP protein is diffusely distributed throughout the cell. (C) Drebrin-YFP expression in COS-7 cells induces F-actin–rich filopodia containing drebrin-YFP (arrows). Drebrin-YFP also localizes to stress fibers (arrowheads). (D and E) CC-YFP (D) or CC-Hel-YFP (E) expression in COS-7 cells also induces F-actin rich filopodia (arrows). The CC-YFP (D) and the CC-Hel-YFP (E) protein localize to filopodia (arrows) and to stress fibers (arrowheads). (F) The CC and Hel domains of drebrin induce filopodia to the same extent as the full-length protein. Shown is a quantification of the number of filopodia per unit length of cell perimeter (filopodia density) in COS-7 cells. Expression of drebrin deletion constructs that contain the CC, the Hel, or both domains, including full-length drebrin, significantly increases the density of filopodia. Surprisingly, filopodia formation induced by the Hel domain is suppressed in the Hel-PP-BB deletion construct. Mock, mock transfection; YFP, YFP alone transfection. Values are mean ± SEM (error bars) of at least 10 cells per transfection from three independent experiments. Significant differences (unpaired Student’s t test): *, P
    Figure Legend Snippet: Drebrin contains two F-actin–binding domains responsible for filopodia formation in heterologous cells. (A) Domain diagrams of drebrin and drebrin deletion constructs used for the functional domain analysis of drebrin. Drebrin has five potential functional domains identified by an in silico analysis using InterPro, Pfam, SMART, and PROSITE. Constructs are YFP-tagged at the C terminus. ADFH, actin-depolymerizing factor homology domain (red, residues 1–135); CC, coiled-coil domain (turquoise, residues 176–256); Hel, helical domain (orange, residues 256–355); PP, proline-rich region (green, residues 364–417); BB, blue box, drebrin C-terminal region (blue, residues 431–649). Numbering is for human drebrin E. (B–E) Immunofluorescence confocal images of COS-7 cells transfected with cDNA encoding YFP-tagged drebrin and drebrin deletion constructs (green) and labeled with phalloidin for F-actin (red). Bars, 5 µm. (B) COS-7 cells expressing YFP alone are rounded and have few processes. The YFP protein is diffusely distributed throughout the cell. (C) Drebrin-YFP expression in COS-7 cells induces F-actin–rich filopodia containing drebrin-YFP (arrows). Drebrin-YFP also localizes to stress fibers (arrowheads). (D and E) CC-YFP (D) or CC-Hel-YFP (E) expression in COS-7 cells also induces F-actin rich filopodia (arrows). The CC-YFP (D) and the CC-Hel-YFP (E) protein localize to filopodia (arrows) and to stress fibers (arrowheads). (F) The CC and Hel domains of drebrin induce filopodia to the same extent as the full-length protein. Shown is a quantification of the number of filopodia per unit length of cell perimeter (filopodia density) in COS-7 cells. Expression of drebrin deletion constructs that contain the CC, the Hel, or both domains, including full-length drebrin, significantly increases the density of filopodia. Surprisingly, filopodia formation induced by the Hel domain is suppressed in the Hel-PP-BB deletion construct. Mock, mock transfection; YFP, YFP alone transfection. Values are mean ± SEM (error bars) of at least 10 cells per transfection from three independent experiments. Significant differences (unpaired Student’s t test): *, P

    Techniques Used: Binding Assay, Construct, Functional Assay, In Silico, Immunofluorescence, Transfection, Labeling, Expressing

    24) Product Images from "Protease degradable electrospun fibrous hydrogels"

    Article Title: Protease degradable electrospun fibrous hydrogels

    Journal: Nature communications

    doi: 10.1038/ncomms7639

    In vitro isotropic MePHA hydrogel degradation 50 μl non-fibrous hydrogels of protease degradable ( a ) and non-degradable ( b ) MePHA equilibrated for 48 hours at 37°C retain their cylindrical shape. Scale bar: 5 mm. ( c , d,e,f ) Quantification of HA release from 2 wt% MePHA hydrogels crosslinked with protease degradable ( c,e ) or non-degradable ( d,f ) methacrylated peptides in 5nM rhMMP-2 ( c,d ), varying concentrations of Type II collagenase ( e,f -listed in units of activity per ml) or TTC buffer. Media (rhMMP-2, collagenase, or TTC buffer) was refreshed every two days to maintain enzyme activity and HA release was quantified by monitoring release of a fluorophore covalently bonded to HA. Error bars represent S.D. (n=3,4). *p
    Figure Legend Snippet: In vitro isotropic MePHA hydrogel degradation 50 μl non-fibrous hydrogels of protease degradable ( a ) and non-degradable ( b ) MePHA equilibrated for 48 hours at 37°C retain their cylindrical shape. Scale bar: 5 mm. ( c , d,e,f ) Quantification of HA release from 2 wt% MePHA hydrogels crosslinked with protease degradable ( c,e ) or non-degradable ( d,f ) methacrylated peptides in 5nM rhMMP-2 ( c,d ), varying concentrations of Type II collagenase ( e,f -listed in units of activity per ml) or TTC buffer. Media (rhMMP-2, collagenase, or TTC buffer) was refreshed every two days to maintain enzyme activity and HA release was quantified by monitoring release of a fluorophore covalently bonded to HA. Error bars represent S.D. (n=3,4). *p

    Techniques Used: In Vitro, Activity Assay

    25) Product Images from "Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α"

    Article Title: Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.181

    Glucocorticoid treatment of primary foetal cardiomyocytes dose-dependently replicates in vivo regulation of cardiac gene expression by glucocorticoid action. Treatment of primary foetal C57BL/6 cardiomyocytes for 24 h with dexamethasone (dex) ( a – d ) or corticosterone (cort) ( e – h ) dose-dependently increased levels of mRNA encoding glucocorticoid-induced leucine zipper (GILZ), MyHC α , atrial natriuretic peptide (ANP) and PGC-1 α . Data were analysed by one-way ANOVA with Bonferroni post-hoc test; * P
    Figure Legend Snippet: Glucocorticoid treatment of primary foetal cardiomyocytes dose-dependently replicates in vivo regulation of cardiac gene expression by glucocorticoid action. Treatment of primary foetal C57BL/6 cardiomyocytes for 24 h with dexamethasone (dex) ( a – d ) or corticosterone (cort) ( e – h ) dose-dependently increased levels of mRNA encoding glucocorticoid-induced leucine zipper (GILZ), MyHC α , atrial natriuretic peptide (ANP) and PGC-1 α . Data were analysed by one-way ANOVA with Bonferroni post-hoc test; * P

    Techniques Used: In Vivo, Expressing, Aqueous Normal-phase Chromatography, Pyrolysis Gas Chromatography

    Corticosterone promotes myofibril maturation in foetal cardiomyocytes in a GR-dependent manner. ( a ) Representative images of control (untreated) primary foetal C57BL/6 cardiomyocytes (left panels), or following 100 nM corticosterone (cort) for 24 h (centre panels). To block GR-mediated effects, cells were pre-treated with 1 μ M RU38486 (RU486) for 30 min prior to addition of 100 nM corticosterone for 24 h (RU486+cort; right panels). Cells were stained with α -actinin (red, Z-discs) and DAPI (blue, nuclei). ( b ) Quantification of the effect of 100 nM cort on myofibrillar structure (top graph, see Materials and Methods for details of the scoring system), sarcomere length (middle graph), and Z-structure/Z-disc width (bottom graph). Data were analysed by one-way ANOVA with Bonferroni's post-hoc test; ** P
    Figure Legend Snippet: Corticosterone promotes myofibril maturation in foetal cardiomyocytes in a GR-dependent manner. ( a ) Representative images of control (untreated) primary foetal C57BL/6 cardiomyocytes (left panels), or following 100 nM corticosterone (cort) for 24 h (centre panels). To block GR-mediated effects, cells were pre-treated with 1 μ M RU38486 (RU486) for 30 min prior to addition of 100 nM corticosterone for 24 h (RU486+cort; right panels). Cells were stained with α -actinin (red, Z-discs) and DAPI (blue, nuclei). ( b ) Quantification of the effect of 100 nM cort on myofibrillar structure (top graph, see Materials and Methods for details of the scoring system), sarcomere length (middle graph), and Z-structure/Z-disc width (bottom graph). Data were analysed by one-way ANOVA with Bonferroni's post-hoc test; ** P

    Techniques Used: Blocking Assay, Staining

    26) Product Images from "The obesity-induced transcriptional regulator TRIP-Br2 mediates visceral fat endoplasmic reticulum stress-induced inflammation"

    Article Title: The obesity-induced transcriptional regulator TRIP-Br2 mediates visceral fat endoplasmic reticulum stress-induced inflammation

    Journal: Nature Communications

    doi: 10.1038/ncomms11378

    Both chemically- and HFD-induced ER stress promote TRIP-Br2 expression specifically in visceral fat. qPCR analysis of TRIP-Br2 or ER stress marker (XBP1s) gene expression in ( a , b ) tissues harvested from mice 18 h after intraperitoneal (i.p.) injection with vehicle (Ctrl, control) or tunicamycin (Tuni; 2.5 mg kg −1 ; n =5 per group replicated thrice); ( c , d ) iWAT or gWAT from mice 6, 12 or 18 h after IP injection with vehicle or Tuni (2.5 mg kg −1 ; n =5 per group replicated twice). ( e ) Western blot analysis of TRIP-Br2, BiP or β-actin (loading control (Ctrl)) in iWAT or gWAT from mice treated for 6, 12 or 18 h with vehicle or Tuni (2.5 mg kg −1 , i.p.). qPCR analysis of TRIP-Br2 or ER stress marker gene expression in ( f ) retroperitoneal (rWAT), perirenal (pWAT) or mesenteric (mWAT) adipose tissues after IP injection with vehicle or Tuni (2.5 mg kg −1 ; n =5 per group replicated thrice); ( g ) gWAT from mice after 12 weeks of CD, HFD or HFD with TUDCA (250 mg kg −1 at 0800 hours and 2000 hours i.p., total 500 mg kg −1 for 15 days; n =5 per group); ( h ) gWAT from mice after 4 or 8 wk of CD or HFD ( n =5 per group); ( i ) SVF or mature adipocytes from mice after 12 wk of CD or HFD ( n =5 per group replicated twice); ( j ) gWAT or iWAT adipocytes differentiated from immortalized gWAT or iWAT preadipocytes after 24 h of vehicle or Tuni (1 μg ml −1 ) treatment ( n =3 replicated twice); ( k ) human adipocytes differentiated from immortalized preadipocytes from lean or obese patients treated with vehicle or Tuni (1 μg ml −1 ) for 24 h ( n =4 replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test or ANOVA, * P
    Figure Legend Snippet: Both chemically- and HFD-induced ER stress promote TRIP-Br2 expression specifically in visceral fat. qPCR analysis of TRIP-Br2 or ER stress marker (XBP1s) gene expression in ( a , b ) tissues harvested from mice 18 h after intraperitoneal (i.p.) injection with vehicle (Ctrl, control) or tunicamycin (Tuni; 2.5 mg kg −1 ; n =5 per group replicated thrice); ( c , d ) iWAT or gWAT from mice 6, 12 or 18 h after IP injection with vehicle or Tuni (2.5 mg kg −1 ; n =5 per group replicated twice). ( e ) Western blot analysis of TRIP-Br2, BiP or β-actin (loading control (Ctrl)) in iWAT or gWAT from mice treated for 6, 12 or 18 h with vehicle or Tuni (2.5 mg kg −1 , i.p.). qPCR analysis of TRIP-Br2 or ER stress marker gene expression in ( f ) retroperitoneal (rWAT), perirenal (pWAT) or mesenteric (mWAT) adipose tissues after IP injection with vehicle or Tuni (2.5 mg kg −1 ; n =5 per group replicated thrice); ( g ) gWAT from mice after 12 weeks of CD, HFD or HFD with TUDCA (250 mg kg −1 at 0800 hours and 2000 hours i.p., total 500 mg kg −1 for 15 days; n =5 per group); ( h ) gWAT from mice after 4 or 8 wk of CD or HFD ( n =5 per group); ( i ) SVF or mature adipocytes from mice after 12 wk of CD or HFD ( n =5 per group replicated twice); ( j ) gWAT or iWAT adipocytes differentiated from immortalized gWAT or iWAT preadipocytes after 24 h of vehicle or Tuni (1 μg ml −1 ) treatment ( n =3 replicated twice); ( k ) human adipocytes differentiated from immortalized preadipocytes from lean or obese patients treated with vehicle or Tuni (1 μg ml −1 ) for 24 h ( n =4 replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test or ANOVA, * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Marker, Mouse Assay, Injection, Western Blot, Binding Assay, Two Tailed Test

    TRIP-Br2 is not regulated by known ER stress-induced transcription factor. qPCR analysis of TRIP-Br2 or ER stress markers gene expression in ( a ) iWAT and gWAT from mice IP injected with vehicle or tunicamycin (2.5 mg kg −1 ) for 18 h ( n =5 per group replicated twice); ( b ) gWAT and iWAT from mice after 12 wk of CD or HFD ( n =5 per group replicated twice); ( c ) 3T3-L1 adipocytes treated with vehicle or tunicamycin (1 μg ml −1 ) for indicated time points ( n =3 per group replicated twice); qPCR analysis of Trip-br2 gene expression in adipocytes stably expressing ( d ) CREB3 ( n =3 per group) or ( e ) CHOP ( n =3 per group). All qPCR data are normalized with TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test, * P
    Figure Legend Snippet: TRIP-Br2 is not regulated by known ER stress-induced transcription factor. qPCR analysis of TRIP-Br2 or ER stress markers gene expression in ( a ) iWAT and gWAT from mice IP injected with vehicle or tunicamycin (2.5 mg kg −1 ) for 18 h ( n =5 per group replicated twice); ( b ) gWAT and iWAT from mice after 12 wk of CD or HFD ( n =5 per group replicated twice); ( c ) 3T3-L1 adipocytes treated with vehicle or tunicamycin (1 μg ml −1 ) for indicated time points ( n =3 per group replicated twice); qPCR analysis of Trip-br2 gene expression in adipocytes stably expressing ( d ) CREB3 ( n =3 per group) or ( e ) CHOP ( n =3 per group). All qPCR data are normalized with TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test, * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Mouse Assay, Injection, Stable Transfection, Binding Assay, Two Tailed Test

    Ablation of TRIP-Br2 abolished ER stress induced-inflammatory and acute phase response in visceral fat. qPCR analysis of inflammation and acute phase reactant markers gene expression in ( a ) gWAT or iWAT fat from C57BL/6 mice treated with vehicle or tunicamycin (Tuni; 2.5 mg kg −1 , i.p.) for 18 h ( n =5 per group replicated twice). ( b ) gWAT from TRIP-Br2 WT or KO mice after 18 h of vehicle or tunicamycin (2.5 mg kg −1 , i.p.) treatment ( n =5 per group replicated twice). ( c ) Plasma cytokine levels in TRIP-Br2 WT or KO mice after 18 h of vehicle or Tuni (2.5 mg kg −1 , i.p.) treatment ( n =5 per group); qPCR analysis of inflammation and acute phase reactant markers gene expression in ( d ) gWAT adipocytes differentiated from primary WT or KO SVF treated with or without Tuni (1 μg ml −1 ) for 24 h ( n =3 per group replicated twice); ( e ) WT gWAT or iWAT adipocytes with or without TRIP-Br2 overexpression ( n =3 per group replicated twice). ( f ) gWAT from WT mice 9 days after injection with Ad-LacZ ( n =6) or Ad-TRIP-Br2 ( n =8) into gWAT in vivo . ( g ) gWAT from WT or KO mice fed with 12 weeks (wk) of HFD ( n =6 per group replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. ( h ) Plasma cytokine levels in WT or KO mice after 12 wk of HFD ( n =5 per group). Data presented as mean±s.e.m. Two-tailed Student's t -test, * P
    Figure Legend Snippet: Ablation of TRIP-Br2 abolished ER stress induced-inflammatory and acute phase response in visceral fat. qPCR analysis of inflammation and acute phase reactant markers gene expression in ( a ) gWAT or iWAT fat from C57BL/6 mice treated with vehicle or tunicamycin (Tuni; 2.5 mg kg −1 , i.p.) for 18 h ( n =5 per group replicated twice). ( b ) gWAT from TRIP-Br2 WT or KO mice after 18 h of vehicle or tunicamycin (2.5 mg kg −1 , i.p.) treatment ( n =5 per group replicated twice). ( c ) Plasma cytokine levels in TRIP-Br2 WT or KO mice after 18 h of vehicle or Tuni (2.5 mg kg −1 , i.p.) treatment ( n =5 per group); qPCR analysis of inflammation and acute phase reactant markers gene expression in ( d ) gWAT adipocytes differentiated from primary WT or KO SVF treated with or without Tuni (1 μg ml −1 ) for 24 h ( n =3 per group replicated twice); ( e ) WT gWAT or iWAT adipocytes with or without TRIP-Br2 overexpression ( n =3 per group replicated twice). ( f ) gWAT from WT mice 9 days after injection with Ad-LacZ ( n =6) or Ad-TRIP-Br2 ( n =8) into gWAT in vivo . ( g ) gWAT from WT or KO mice fed with 12 weeks (wk) of HFD ( n =6 per group replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. ( h ) Plasma cytokine levels in WT or KO mice after 12 wk of HFD ( n =5 per group). Data presented as mean±s.e.m. Two-tailed Student's t -test, * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Mouse Assay, Over Expression, Injection, In Vivo, Binding Assay, Two Tailed Test

    ER stress induces GATA3 expression in visceral adipocytes and leads to TRIP-Br2 upregulation. qPCR analysis of GATA1, GATA2 or GATA3 gene expression in ( a ) 3T3-L1 differentiated adipocytes ( n =3 per group replicated twice); ( b ) WT1 differentiated adipocytes ( n =3 per group replicated twice) after 6 h of vehicle or tunicamycin (Tuni; 1 μg ml −1 ) treatment; ( c ) WT gWAT or iWAT ( n =5 per group replicated twice) 18 h after vehicle or Tuni (2.5 mg kg −1 , i.p.) injection; ( d ) WT gWAT or iWAT adipocytes ( n =3 per group) after vehicle or Tuni (1 μg ml −1 ) treatment for 6 h; ( e ) gWAT and iWAT SVF or mature adipocytes after 12 weeks of CD or HFD ( n =5 per group replicated twice); ( f ) gWAT and iWAT from lean or obese ob/ob mice ( n =6 per group); ( g ) human adipocytes from immortalized human preadipocytes or SGBS cells after vehicle or Tuni (1 μg ml −1 ) treatment for 24 h ( n =3–4 per group replicated twice). ( h ) Gene expression of human TRIP-Br2 and GATA3 in visceral adipose tissue from human subjects ( n =24 per group). qPCR analysis of TRIP-Br2 in ( i ) gWAT adipocytes treated with Tuni (1 μg ml −1 ) and/or GATA inhibitor K7174 (1, 5 μM) for 24 h ( n =3 per group replicated twice); ( j ) gWAT adipocytes infected with control (Ctrl) or DN-GATA3 retrovirus ( n =3 per group); ( k ) gWAT adipocytes infected with Ctrl or GATA3 retrovirus ( n =3 per group replicated twice); ( l ) gWAT adipocytes differentiated from GATA3fl/fl-CreERT2 primary SVF with or without tamoxifen (0.5 μM) treatment to induce GATA3 KO ( n =5 per group). ( m ) qPCR analysis of proximal or distal genomic region of TRIP-Br2 transcript 3 promoter after GATA3 chromatin immunoprecipitation ( n =3 per group replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test or ANOVA, * P
    Figure Legend Snippet: ER stress induces GATA3 expression in visceral adipocytes and leads to TRIP-Br2 upregulation. qPCR analysis of GATA1, GATA2 or GATA3 gene expression in ( a ) 3T3-L1 differentiated adipocytes ( n =3 per group replicated twice); ( b ) WT1 differentiated adipocytes ( n =3 per group replicated twice) after 6 h of vehicle or tunicamycin (Tuni; 1 μg ml −1 ) treatment; ( c ) WT gWAT or iWAT ( n =5 per group replicated twice) 18 h after vehicle or Tuni (2.5 mg kg −1 , i.p.) injection; ( d ) WT gWAT or iWAT adipocytes ( n =3 per group) after vehicle or Tuni (1 μg ml −1 ) treatment for 6 h; ( e ) gWAT and iWAT SVF or mature adipocytes after 12 weeks of CD or HFD ( n =5 per group replicated twice); ( f ) gWAT and iWAT from lean or obese ob/ob mice ( n =6 per group); ( g ) human adipocytes from immortalized human preadipocytes or SGBS cells after vehicle or Tuni (1 μg ml −1 ) treatment for 24 h ( n =3–4 per group replicated twice). ( h ) Gene expression of human TRIP-Br2 and GATA3 in visceral adipose tissue from human subjects ( n =24 per group). qPCR analysis of TRIP-Br2 in ( i ) gWAT adipocytes treated with Tuni (1 μg ml −1 ) and/or GATA inhibitor K7174 (1, 5 μM) for 24 h ( n =3 per group replicated twice); ( j ) gWAT adipocytes infected with control (Ctrl) or DN-GATA3 retrovirus ( n =3 per group); ( k ) gWAT adipocytes infected with Ctrl or GATA3 retrovirus ( n =3 per group replicated twice); ( l ) gWAT adipocytes differentiated from GATA3fl/fl-CreERT2 primary SVF with or without tamoxifen (0.5 μM) treatment to induce GATA3 KO ( n =5 per group). ( m ) qPCR analysis of proximal or distal genomic region of TRIP-Br2 transcript 3 promoter after GATA3 chromatin immunoprecipitation ( n =3 per group replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test or ANOVA, * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Injection, Mouse Assay, Infection, Chromatin Immunoprecipitation, Binding Assay, Two Tailed Test

    27) Product Images from "Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice"

    Article Title: Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice

    Journal: Molecular Pain

    doi: 10.1177/1744806916666284

    GM3S depletion prevents increased intracellular calcium influx in DRG neurons in diet-induced diabetes. (a–d) Representative tracings of intracellular calcium responses ([Ca++] i ) to different concentration of potassium (K + ) (10–50 mM) of acutely cultured DRG sensory neurons from control WT RD (a, n = 56), WT HFD (b, n = 66), GM3S KO RD (c, n = 108), and GM3S KO HFD (d, n = 41) mice. (e) Percentage of DRG neurons from each group responding to different concentration of potassium (K + ) (10, 25, and 50 mM). Values are expressed as mean ± SE. *** p
    Figure Legend Snippet: GM3S depletion prevents increased intracellular calcium influx in DRG neurons in diet-induced diabetes. (a–d) Representative tracings of intracellular calcium responses ([Ca++] i ) to different concentration of potassium (K + ) (10–50 mM) of acutely cultured DRG sensory neurons from control WT RD (a, n = 56), WT HFD (b, n = 66), GM3S KO RD (c, n = 108), and GM3S KO HFD (d, n = 41) mice. (e) Percentage of DRG neurons from each group responding to different concentration of potassium (K + ) (10, 25, and 50 mM). Values are expressed as mean ± SE. *** p

    Techniques Used: Concentration Assay, Cell Culture, Mouse Assay

    28) Product Images from "Sleep Fragmentation Promotes NADPH Oxidase 2-Mediated Adipose Tissue Inflammation Leading to Insulin Resistance in Mice"

    Article Title: Sleep Fragmentation Promotes NADPH Oxidase 2-Mediated Adipose Tissue Inflammation Leading to Insulin Resistance in Mice

    Journal: International journal of obesity (2005)

    doi: 10.1038/ijo.2013.139

    SF-induced increases in global macrophage numbers in visceral fat of wild-type, but not Nox2-deficient mice A. A representative flow cytometry analysis showing a moderate increase in the number of F4/80 + CD11b + macrophages in visceral fat of a WT mouse exposed to SF for 2.5 weeks. This phenomenon was absent in Nox2-deficient gp91 phox-/Y mice. B. Summary of 4 independent flow cytometry experiments. Data are mean ± SE; n=4 for each group.
    Figure Legend Snippet: SF-induced increases in global macrophage numbers in visceral fat of wild-type, but not Nox2-deficient mice A. A representative flow cytometry analysis showing a moderate increase in the number of F4/80 + CD11b + macrophages in visceral fat of a WT mouse exposed to SF for 2.5 weeks. This phenomenon was absent in Nox2-deficient gp91 phox-/Y mice. B. Summary of 4 independent flow cytometry experiments. Data are mean ± SE; n=4 for each group.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    SF-induced selective increase in the number of pro-inflammatory M1 macrophages in visceral fat of wild-type, but not Nox2-deficient mice A. A representative flow cytometry analysis showing an increase in the number of CD11c + M1 macrophages in visceral fat of a WT mouse exposed to SF for 2.5 weeks. Such SF-induced effect on the pro-inflammatory M1 subset was not observed in Nox2-deficient gp91 phox-/Y mice. B. Summary of 5 independent flow cytometry experiments. Notice that SF-induced decrease in the M2 subset was not significant at this stage of chronic SF. Data are mean ± SE; n=5 for each group.
    Figure Legend Snippet: SF-induced selective increase in the number of pro-inflammatory M1 macrophages in visceral fat of wild-type, but not Nox2-deficient mice A. A representative flow cytometry analysis showing an increase in the number of CD11c + M1 macrophages in visceral fat of a WT mouse exposed to SF for 2.5 weeks. Such SF-induced effect on the pro-inflammatory M1 subset was not observed in Nox2-deficient gp91 phox-/Y mice. B. Summary of 5 independent flow cytometry experiments. Notice that SF-induced decrease in the M2 subset was not significant at this stage of chronic SF. Data are mean ± SE; n=5 for each group.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    29) Product Images from "Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture"

    Article Title: Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

    Journal: Nature medicine

    doi: 10.1038/nm.3585

    Systematic evaluation of oncogene modules of increasing complexity in primary colon organoids (a,b) In vitro organoid Apc deletion. Primary small intestine (a) or colon (b) organoids from neonatal Apc flox/flox ; villin-CreER mice were treated with tamoxifen (2 μM, 7 days) followed by analysis at day 20. Tamoxifen-mediated Apc deletion induced overall growth, proliferative index (middle, PCNA) and APC deletion (bottom, APC IHC). Scale bars, top, 5 mm; middle, 50 μm; bottom, 50 μm. (c,d) In situ polyposis within long-term intestinal organoid cultures. Small intestine organoids from neonatal Apc flox/flox ; villin-CreER mice induced without (c) or with (d) tamoxifen followed by culture for 105 days and analysis by PCNA. An in situ tubular adenomatous polyp within the organoid wall in (d) exhibits a polypoid proliferation of PCNA-positive closely spaced tubules lined by enlarged, crowded nuclei. H E staining of the yellow-boxed region is depicted. Scale bars, 50 μm. (e) one to four oncogene CRC modules were created by infection of tamoxifen-treated Apc flox/flox ; villin-CreER adult colon organoids with appropriate combinations of control LMP retrovirus (with GFP cassette) or retroviruses encoding Kras G12D , LMP p53 shRNA/GFP or LMP Smad4 shRNA/GFP followed by H E staining at day 50 post-infection. The 1-gene Apc (“A”) and 2-gene colon modules ( Apc −/− /Kras G12D (AK), Apc −/− /p53 shRNA (AP) or Apc −/− /Smad4 shRNA (AS)) only exhibited minimal dysplasia. High-grade focal dysplasia was exhibited by the 3-gene module Apc −/− /Kras G12D /p53 shRNA (AKP). The four-gene module Apc −/− /Kras G12D /p53 shRNA/ Smad4 shRNA (AKPS) induced adenocarcinoma characterized by atypia, confluent sheets of cells, cribriform growth patterns, luminal necrosis, jagged infiltrating growth patterns and frank invasion. Scale bars, 50 μm.
    Figure Legend Snippet: Systematic evaluation of oncogene modules of increasing complexity in primary colon organoids (a,b) In vitro organoid Apc deletion. Primary small intestine (a) or colon (b) organoids from neonatal Apc flox/flox ; villin-CreER mice were treated with tamoxifen (2 μM, 7 days) followed by analysis at day 20. Tamoxifen-mediated Apc deletion induced overall growth, proliferative index (middle, PCNA) and APC deletion (bottom, APC IHC). Scale bars, top, 5 mm; middle, 50 μm; bottom, 50 μm. (c,d) In situ polyposis within long-term intestinal organoid cultures. Small intestine organoids from neonatal Apc flox/flox ; villin-CreER mice induced without (c) or with (d) tamoxifen followed by culture for 105 days and analysis by PCNA. An in situ tubular adenomatous polyp within the organoid wall in (d) exhibits a polypoid proliferation of PCNA-positive closely spaced tubules lined by enlarged, crowded nuclei. H E staining of the yellow-boxed region is depicted. Scale bars, 50 μm. (e) one to four oncogene CRC modules were created by infection of tamoxifen-treated Apc flox/flox ; villin-CreER adult colon organoids with appropriate combinations of control LMP retrovirus (with GFP cassette) or retroviruses encoding Kras G12D , LMP p53 shRNA/GFP or LMP Smad4 shRNA/GFP followed by H E staining at day 50 post-infection. The 1-gene Apc (“A”) and 2-gene colon modules ( Apc −/− /Kras G12D (AK), Apc −/− /p53 shRNA (AP) or Apc −/− /Smad4 shRNA (AS)) only exhibited minimal dysplasia. High-grade focal dysplasia was exhibited by the 3-gene module Apc −/− /Kras G12D /p53 shRNA (AKP). The four-gene module Apc −/− /Kras G12D /p53 shRNA/ Smad4 shRNA (AKPS) induced adenocarcinoma characterized by atypia, confluent sheets of cells, cribriform growth patterns, luminal necrosis, jagged infiltrating growth patterns and frank invasion. Scale bars, 50 μm.

    Techniques Used: In Vitro, Mouse Assay, Immunohistochemistry, In Situ, Staining, Infection, shRNA, ALP Assay

    30) Product Images from "BIN1 Localizes the L-Type Calcium Channel to Cardiac T-TubulesBIN1: A Protein with Great Heart"

    Article Title: BIN1 Localizes the L-Type Calcium Channel to Cardiac T-TubulesBIN1: A Protein with Great Heart

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1000312

    BIN1 knockdown delays calcium transient development in mouse cardiomyocytes. (A) Western blot indicates an 80% knockdown of BIN1 protein by siRNA in differentiated mouse cardiomyocytes. (B) Surface biotinylation of Cav1.2 in these primary cardiomyocytes indicates a 45% reduction of surface Cav1.2 after BIN1 knockdown. (C) Live cell calcium imaging in differentiated cardiomyocytes indicates that BIN1 knockdown also delays calcium transient development in these cells. Average time to 50% maximal fluorescence intensity (T1/2 max) of calcium transient is presented in the left panel (* p
    Figure Legend Snippet: BIN1 knockdown delays calcium transient development in mouse cardiomyocytes. (A) Western blot indicates an 80% knockdown of BIN1 protein by siRNA in differentiated mouse cardiomyocytes. (B) Surface biotinylation of Cav1.2 in these primary cardiomyocytes indicates a 45% reduction of surface Cav1.2 after BIN1 knockdown. (C) Live cell calcium imaging in differentiated cardiomyocytes indicates that BIN1 knockdown also delays calcium transient development in these cells. Average time to 50% maximal fluorescence intensity (T1/2 max) of calcium transient is presented in the left panel (* p

    Techniques Used: Western Blot, Imaging, Fluorescence

    BIN1 colocalizes with Cav1.2 at T-tubules in cardiomyocytes. (A) Confocal image (100×) of human (left) and mouse (right) adult cardiomyocytes. The cells were fixed and stained with mouse anti-BIN1 or rabbit anti-Cav1.2. Two-dimensional frames of Cav1.2 and BIN1 are shown in the top panel. Cardiomyocyte fluorescence intensity profiles along the cardiomyocyte longitudinal axis are presented in the middle panel. The bottom panel is the power spectrum over spatial distance averaged from five cardiomyocytes, which indicate that both BIN1 and Cav1.2 signals occurs at every 2 µm (fundamental peak occurs at ∼2 µm). Note the small peak at 1 µm is a harmonic of the fundamental peak at 2 µm (scale bar: 10 µm). (B) Confocal images (100×) of human (left) and mouse (right) cardiomyocytes stained with mouse anti-BIN1 (green) and rabbit anti-Cav1.2 (red) reveal colocalization between BIN1 and Cav1.2 along T-tubules (scale bar: 5 µm). Pearson colocalization coefficient and scatter plot between BIN1 and Cav1.2 are also shown in this panel. (C) Electron microscopy image of adult mouse cardiomyocytes fixed and immunogold labeled for BIN1 (small dots) and Cav1.2 (large dots) (scale bar: 200 nm) (left). As seen in the enlarged image, BIN1 and Cav1.2 occurs within 50 nm on T-tubule membranes. The negative control image without primary antibodies incubation is shown at the right panel.
    Figure Legend Snippet: BIN1 colocalizes with Cav1.2 at T-tubules in cardiomyocytes. (A) Confocal image (100×) of human (left) and mouse (right) adult cardiomyocytes. The cells were fixed and stained with mouse anti-BIN1 or rabbit anti-Cav1.2. Two-dimensional frames of Cav1.2 and BIN1 are shown in the top panel. Cardiomyocyte fluorescence intensity profiles along the cardiomyocyte longitudinal axis are presented in the middle panel. The bottom panel is the power spectrum over spatial distance averaged from five cardiomyocytes, which indicate that both BIN1 and Cav1.2 signals occurs at every 2 µm (fundamental peak occurs at ∼2 µm). Note the small peak at 1 µm is a harmonic of the fundamental peak at 2 µm (scale bar: 10 µm). (B) Confocal images (100×) of human (left) and mouse (right) cardiomyocytes stained with mouse anti-BIN1 (green) and rabbit anti-Cav1.2 (red) reveal colocalization between BIN1 and Cav1.2 along T-tubules (scale bar: 5 µm). Pearson colocalization coefficient and scatter plot between BIN1 and Cav1.2 are also shown in this panel. (C) Electron microscopy image of adult mouse cardiomyocytes fixed and immunogold labeled for BIN1 (small dots) and Cav1.2 (large dots) (scale bar: 200 nm) (left). As seen in the enlarged image, BIN1 and Cav1.2 occurs within 50 nm on T-tubule membranes. The negative control image without primary antibodies incubation is shown at the right panel.

    Techniques Used: Staining, Fluorescence, Electron Microscopy, Labeling, Negative Control, Incubation

    Antegrade trafficking of Cav1.2 is microtubule dependent. (A) Surface biotinylation of adult mouse cardiomyocytes indicates that nocodazole (30 µM) progressively reduces surface Cav1.2 expression in the presence of an endocytosis inhibitor dynasore (20 µM). Note that dynasore alone significantly increases surface expression of Cav1.2 by blocking dynamin-dependent endocytosis of Cav1.2 in cardiomyocytes. (B) Top panel: Confocal images (100×) of mouse cardiomyocytes stained with rabbit anti-Cav1.2 (red) and mouse anti-α-tubulin (green) reveal localization of Cav1.2 on microtubule network (scale bar: 5 µm). Bottom panel: Deconvolution of wide-field image of HL-1 cells stained with Cav1.2 (red) and α-tubulin (green). Merged image shows localization of Cav1.2 to the microtubule network. Enlarged pictures (right) indicate that Cav1.2 is distributed along microtubules (## p
    Figure Legend Snippet: Antegrade trafficking of Cav1.2 is microtubule dependent. (A) Surface biotinylation of adult mouse cardiomyocytes indicates that nocodazole (30 µM) progressively reduces surface Cav1.2 expression in the presence of an endocytosis inhibitor dynasore (20 µM). Note that dynasore alone significantly increases surface expression of Cav1.2 by blocking dynamin-dependent endocytosis of Cav1.2 in cardiomyocytes. (B) Top panel: Confocal images (100×) of mouse cardiomyocytes stained with rabbit anti-Cav1.2 (red) and mouse anti-α-tubulin (green) reveal localization of Cav1.2 on microtubule network (scale bar: 5 µm). Bottom panel: Deconvolution of wide-field image of HL-1 cells stained with Cav1.2 (red) and α-tubulin (green). Merged image shows localization of Cav1.2 to the microtubule network. Enlarged pictures (right) indicate that Cav1.2 is distributed along microtubules (## p

    Techniques Used: Expressing, Blocking Assay, Staining

    31) Product Images from "Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice"

    Article Title: Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt52

    A single-vector tetracycline construct allows doxycycline-regulated expression . (A) Overview showing the regulator plasmid containing the EF1α-tTA cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the TetO-mCh-Rs1 cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to
    Figure Legend Snippet: A single-vector tetracycline construct allows doxycycline-regulated expression . (A) Overview showing the regulator plasmid containing the EF1α-tTA cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the TetO-mCh-Rs1 cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to "leakiness" or steric interference from endogenous promoter activity. (B, C) HEK-293 cells carrying the Exp-pcDNA3.2(EF1α-tTA/TetO-mCh-Rs1) expression cassette and cultured in doxycycline (suppressed expression) or in the absence of doxycycline (transgene expression allowed) demonstrate doxycycline-dependent mCherry expression. (D) Schematic of targeted Rosa26 locus and Southern screening strategy. The Rosa26 locus in E14 ES cells was targeted by homologous recombination with the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. Regions in hatch marks indicate the 5' and 3' homology regions of the targeting vector and the endogenous Rosa26 locus (abbreviated R26 in the figure). The location of the 5' recombination Southern probe and HindIII restriction sites are indicated. (E) Southern blots of genomic DNA digested with HindIII and probed as in (D). Heterozygous ES cells at the Rosa26 locus are indicated by the two bands.

    Techniques Used: Plasmid Preparation, Construct, Expressing, Sequencing, Activation Assay, Activity Assay, Cell Culture, Homologous Recombination

    A single copy of the EF1α-tTA regulator region weakly drives expression of a TetO transgene in mice . (A) Areal bone mineral density by DEXA of nine-week-old mice shows that the ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice have increased bone mass. N = 9 WT, 5 R26(EF1α-tTA/TetO-mCh-Rs1), and 9 ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice. ***, P
    Figure Legend Snippet: A single copy of the EF1α-tTA regulator region weakly drives expression of a TetO transgene in mice . (A) Areal bone mineral density by DEXA of nine-week-old mice shows that the ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice have increased bone mass. N = 9 WT, 5 R26(EF1α-tTA/TetO-mCh-Rs1), and 9 ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice. ***, P

    Techniques Used: Expressing, Mouse Assay

    32) Product Images from "Novel Methodology for Creating Macaque Retinas with Sortable Photoreceptors and Ganglion Cells"

    Article Title: Novel Methodology for Creating Macaque Retinas with Sortable Photoreceptors and Ganglion Cells

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2016.00551

    Details of tissue processing from OD and OS eyes of animal AV263 . Anterior segment and vitreous were removed from both eyes and eyecups immersed in oxygenated Ames media. Retinas were carefully isolated from RPE. The OS retina was divided into four quadrants, each intended for purposes described within the top panel . During incubation in Ames media, optic nerve from the OD eyecup was incubated in 2.5 mg/mL micro-ruby™ diluted in oxygenated Ames media for 3 h at room temperature. The OD retina was then divided into four quadrants, each intended for purposes described within the bottom panel .
    Figure Legend Snippet: Details of tissue processing from OD and OS eyes of animal AV263 . Anterior segment and vitreous were removed from both eyes and eyecups immersed in oxygenated Ames media. Retinas were carefully isolated from RPE. The OS retina was divided into four quadrants, each intended for purposes described within the top panel . During incubation in Ames media, optic nerve from the OD eyecup was incubated in 2.5 mg/mL micro-ruby™ diluted in oxygenated Ames media for 3 h at room temperature. The OD retina was then divided into four quadrants, each intended for purposes described within the bottom panel .

    Techniques Used: Isolation, Incubation

    33) Product Images from "SMAD3 Deficiency Promotes Inflammatory Aortic Aneurysms in Angiotensin II-Infused Mice Via Activation of iNOS"

    Article Title: SMAD3 Deficiency Promotes Inflammatory Aortic Aneurysms in Angiotensin II-Infused Mice Via Activation of iNOS

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.113.000269

    Defective contractile function of S3KO aorta. A through C, PE‐induced contraction of WT and S3KO aortic rings treated with placebo (A), 200 μmol/L l‐NAME (B), or 200 μmol/L aminoguanidine (C) (n=4 per group; duplicate experiments were performed). Contraction was measured at PE concentrations ranging from 10 −10 to 10 −3 mol/L, and concentration–response curves were constructed. Note that contraction is expressed as the percentage of maximal contraction induced by incubation of aortic rings in 80 mmol/L high potassium (Hi‐K + ) buffer. For placebo and l‐NAME treatment, endothelium‐intact aortic segments were used, whereas endothelium‐denuded aortas were used in aminoguanidine treatment to eliminate the vasorelaxant effect of eNOS‐derived NO. D, pEC 50 of WT and S3KO aortas in response to PE, indicating tissue sensitivity toward the drug. Note that pEC 50 is the negative logarithm of EC 50 , which refers to the concentration of a drug that induces a half‐maximal response. E, Maximal contraction force generated when incubated with 80 mmol/L Hi‐K + buffer. F, Aortic mRNA level of AngII type 1a (AT1a), type 1b (AT1b), and type 2 (AT2) receptors in WT and S3KO mice (n=3 per group; triplicate experiments were performed). Gene expression was normalized to RPL27. G through I, AngII‐induced contraction in ex vivo aortic segments treated with placebo (G), 200 μmol/L l‐NAME (H), or 200 μmol/L aminoguanidine (I) (n=5 per group; duplicate experiments were performed). Note that AngII elicited a rapid and transient contraction of infrarenal aortic segments at a concentration of 1 μg/mL, whereas minimal (
    Figure Legend Snippet: Defective contractile function of S3KO aorta. A through C, PE‐induced contraction of WT and S3KO aortic rings treated with placebo (A), 200 μmol/L l‐NAME (B), or 200 μmol/L aminoguanidine (C) (n=4 per group; duplicate experiments were performed). Contraction was measured at PE concentrations ranging from 10 −10 to 10 −3 mol/L, and concentration–response curves were constructed. Note that contraction is expressed as the percentage of maximal contraction induced by incubation of aortic rings in 80 mmol/L high potassium (Hi‐K + ) buffer. For placebo and l‐NAME treatment, endothelium‐intact aortic segments were used, whereas endothelium‐denuded aortas were used in aminoguanidine treatment to eliminate the vasorelaxant effect of eNOS‐derived NO. D, pEC 50 of WT and S3KO aortas in response to PE, indicating tissue sensitivity toward the drug. Note that pEC 50 is the negative logarithm of EC 50 , which refers to the concentration of a drug that induces a half‐maximal response. E, Maximal contraction force generated when incubated with 80 mmol/L Hi‐K + buffer. F, Aortic mRNA level of AngII type 1a (AT1a), type 1b (AT1b), and type 2 (AT2) receptors in WT and S3KO mice (n=3 per group; triplicate experiments were performed). Gene expression was normalized to RPL27. G through I, AngII‐induced contraction in ex vivo aortic segments treated with placebo (G), 200 μmol/L l‐NAME (H), or 200 μmol/L aminoguanidine (I) (n=5 per group; duplicate experiments were performed). Note that AngII elicited a rapid and transient contraction of infrarenal aortic segments at a concentration of 1 μg/mL, whereas minimal (

    Techniques Used: Concentration Assay, Construct, Incubation, Derivative Assay, Generated, Mouse Assay, Expressing, Ex Vivo

    34) Product Images from "Cardiac O-GlcNAcylation blunts autophagic signaling in the diabetic heart"

    Article Title: Cardiac O-GlcNAcylation blunts autophagic signaling in the diabetic heart

    Journal: Life sciences

    doi: 10.1016/j.lfs.2012.06.011

    The role of the hexosamine biosynthesis pathway (HBP) in blunting autophagic signaling. Representative western blots showing Beclin-1 and LC3 protein in cardiomyocytes from non-diabetic (Con) and type 2 diabetic ( db/db ) under basal conditions and in response
    Figure Legend Snippet: The role of the hexosamine biosynthesis pathway (HBP) in blunting autophagic signaling. Representative western blots showing Beclin-1 and LC3 protein in cardiomyocytes from non-diabetic (Con) and type 2 diabetic ( db/db ) under basal conditions and in response

    Techniques Used: Western Blot

    The temporal progression of body weight (A) and survival (B) following either sham or trans-aortic constriction surgery to induce a pressure overload (PO) in non-diabetic (control), high fat-fed, or high fat-fed and STZ treated type 2 diabetic rats. †
    Figure Legend Snippet: The temporal progression of body weight (A) and survival (B) following either sham or trans-aortic constriction surgery to induce a pressure overload (PO) in non-diabetic (control), high fat-fed, or high fat-fed and STZ treated type 2 diabetic rats. †

    Techniques Used:

    35) Product Images from "The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair"

    Article Title: The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair

    Journal: Journal of Clinical Investigation

    doi:

    SMC migration was reduced in the absence of DDR1. SMCs from DDR1+/+ mice (filled bars) or DDR1–/– mice (open bars) were stimulated to migrate for 4 hours in chemotaxis chambers with 200 nM type I collagen (left) or type VIII collagen (right) added to DMEM in the bottom of the chamber. Migration was quantified by staining and counting the number of cells that migrated to the bottom of the filter. Experiments were done in triplicate and repeated three times. A Values from DDR1–/– SMCs were significantly less than values from wild-type SMCs.
    Figure Legend Snippet: SMC migration was reduced in the absence of DDR1. SMCs from DDR1+/+ mice (filled bars) or DDR1–/– mice (open bars) were stimulated to migrate for 4 hours in chemotaxis chambers with 200 nM type I collagen (left) or type VIII collagen (right) added to DMEM in the bottom of the chamber. Migration was quantified by staining and counting the number of cells that migrated to the bottom of the filter. Experiments were done in triplicate and repeated three times. A Values from DDR1–/– SMCs were significantly less than values from wild-type SMCs.

    Techniques Used: Migration, Mouse Assay, Chemotaxis Assay, Staining

    Cell proliferation on collagen was less in DDR1–/– SMCs compared with DDR1+/+ SMCs. ( a ) SMCs from DDR1+/+ mice (filled bars) or DDR1–/– mice (open bars) were stimulated to grow with 10% FCS and incubated with [ 3 H]thymidine on wells coated with 100 nM type I collagen (left) or type VIII collagen (right). A Value from DDR1–/– SMCs was significantly less than value from wild-type SMCs. ( b ) Number of SMCs per well after plating on 100 nM type I collagen and growing in DMEM containing 10% FCS for 1–4 days. Squares, DDR1+/+ SMCs; circles, DDR1–/– SMCs. Experiments were done in triplicate and repeated three times. A Value from DDR1–/– SMCs was significantly less than value from wild-type SMCs.
    Figure Legend Snippet: Cell proliferation on collagen was less in DDR1–/– SMCs compared with DDR1+/+ SMCs. ( a ) SMCs from DDR1+/+ mice (filled bars) or DDR1–/– mice (open bars) were stimulated to grow with 10% FCS and incubated with [ 3 H]thymidine on wells coated with 100 nM type I collagen (left) or type VIII collagen (right). A Value from DDR1–/– SMCs was significantly less than value from wild-type SMCs. ( b ) Number of SMCs per well after plating on 100 nM type I collagen and growing in DMEM containing 10% FCS for 1–4 days. Squares, DDR1+/+ SMCs; circles, DDR1–/– SMCs. Experiments were done in triplicate and repeated three times. A Value from DDR1–/– SMCs was significantly less than value from wild-type SMCs.

    Techniques Used: Mouse Assay, Incubation

    36) Product Images from "Endothelial MT1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis"

    Article Title: Endothelial MT1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201910862

    Identification of TSP 1 cleavage sites for MT 1‐ MMP Western blot of TSP1 expression (developed with an antibody against an epitope nearby the N‐terminus; Lee et al , 2006 ) in lysates from siCtrl and siMT1‐silenced HUVEC. MT1‐MMP and tubulin are included as silencing and loading controls, respectively. The arrowhead marks full‐length TSP1 and the asterisk the N‐terminal TSP1 fragment. Western blot of in vitro digested TSP1 (developed with the same antibody as in A) incubated with increasing amounts of recombinant human MT1‐MMP catalytic domain. rhMT1‐MMP catalytic domain is also included. The arrowhead marks full‐length TSP1 and the asterisk the N‐terminal TSP1 fragment generated by MT1‐MMP cleavage. In silico model of the membrane‐anchored MT1‐MMP dimer (blue/orange) and TSP1 type 1 repeat domains 2 and 3 (green). Yellow marks the catalytic pocket in the MT1‐MMP protease, and red indicates the two selected cleavage sites in TSP1. Scheme depicting the TSP1 domain structure with the binding sequences to CD36, CD47, and αvβ3 integrin, as well as the positions of the identified cleavage sites for MT1‐MMP. DAF‐FM mean fluorescence intensity (MFI) in HUVEC expressing MT1‐MMP siRNA and left untreated or treated with 200 ng of full‐length TSP1 or the E123CaG‐1 fragment; n = 128–184 cells analyzed per condition in two independent experiments. Data are shown as individual cell values and mean ± SEM and were tested by the Kruskal–Wallis test; * P
    Figure Legend Snippet: Identification of TSP 1 cleavage sites for MT 1‐ MMP Western blot of TSP1 expression (developed with an antibody against an epitope nearby the N‐terminus; Lee et al , 2006 ) in lysates from siCtrl and siMT1‐silenced HUVEC. MT1‐MMP and tubulin are included as silencing and loading controls, respectively. The arrowhead marks full‐length TSP1 and the asterisk the N‐terminal TSP1 fragment. Western blot of in vitro digested TSP1 (developed with the same antibody as in A) incubated with increasing amounts of recombinant human MT1‐MMP catalytic domain. rhMT1‐MMP catalytic domain is also included. The arrowhead marks full‐length TSP1 and the asterisk the N‐terminal TSP1 fragment generated by MT1‐MMP cleavage. In silico model of the membrane‐anchored MT1‐MMP dimer (blue/orange) and TSP1 type 1 repeat domains 2 and 3 (green). Yellow marks the catalytic pocket in the MT1‐MMP protease, and red indicates the two selected cleavage sites in TSP1. Scheme depicting the TSP1 domain structure with the binding sequences to CD36, CD47, and αvβ3 integrin, as well as the positions of the identified cleavage sites for MT1‐MMP. DAF‐FM mean fluorescence intensity (MFI) in HUVEC expressing MT1‐MMP siRNA and left untreated or treated with 200 ng of full‐length TSP1 or the E123CaG‐1 fragment; n = 128–184 cells analyzed per condition in two independent experiments. Data are shown as individual cell values and mean ± SEM and were tested by the Kruskal–Wallis test; * P

    Techniques Used: Western Blot, Expressing, In Vitro, Incubation, Recombinant, Generated, In Silico, Binding Assay, Fluorescence

    37) Product Images from "Domain of Dentine Sialoprotein Mediates Proliferation and Differentiation of Human Periodontal Ligament Stem Cells"

    Article Title: Domain of Dentine Sialoprotein Mediates Proliferation and Differentiation of Human Periodontal Ligament Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081655

    Product of DSP protein coated on glass slides. A . Coomassie-stained SDS-PAGE gel for analysis of expression of the recombinant COOH-terminal dentin sialoprotein (rC-DSP) protein. The sample in DL21 cells was grown 3 h with 1 mM IPTG induction after reaching O.D.600 of 0.6. The rC-DSP was purified by the column described in the materials and methods. B . Expression of purified rC-DSP was confirmed by Western blotting analysis using goat polyclonal anti-DSP antibody (Santa Cruz Biotechnology Inc.). D - E . Collagen type I and rC-DSP proteins were coated on glass slides for overnight at 37°C. For scanning electron microscopy, the samples were washed and dried as well as treated with sodium deoxycholate. Then the samples were observed under scanning electron microscope (SEM). Fibril matrix was visible on the collagen type I coated slide ( D ) and rC-DSP coated slide ( E ), but no control slide ( C ). Also, there were different morphologies between rC-DSP and collagen type I.
    Figure Legend Snippet: Product of DSP protein coated on glass slides. A . Coomassie-stained SDS-PAGE gel for analysis of expression of the recombinant COOH-terminal dentin sialoprotein (rC-DSP) protein. The sample in DL21 cells was grown 3 h with 1 mM IPTG induction after reaching O.D.600 of 0.6. The rC-DSP was purified by the column described in the materials and methods. B . Expression of purified rC-DSP was confirmed by Western blotting analysis using goat polyclonal anti-DSP antibody (Santa Cruz Biotechnology Inc.). D - E . Collagen type I and rC-DSP proteins were coated on glass slides for overnight at 37°C. For scanning electron microscopy, the samples were washed and dried as well as treated with sodium deoxycholate. Then the samples were observed under scanning electron microscope (SEM). Fibril matrix was visible on the collagen type I coated slide ( D ) and rC-DSP coated slide ( E ), but no control slide ( C ). Also, there were different morphologies between rC-DSP and collagen type I.

    Techniques Used: Staining, SDS Page, Expressing, Recombinant, Purification, Western Blot, Electron Microscopy, Microscopy

    38) Product Images from "Primary Culture of Mammalian Taste Epithelium"

    Article Title: Primary Culture of Mammalian Taste Epithelium

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-62703-125-7_7

    Attachment and morphology of cultured human fungiform taste cells. ( a ) Primary cell cultures grown on collagen type-1-coated plates were imaged after 2 days. ( b ) Cells from human fungiform papillae grew for up to 2–4 weeks under attached cell clusters. ( c and d ) Represent day 2 and 4 weeks after harvesting, respectively. During this period we did not observe growth of cells with the appearance of non-taste epithelial cells.
    Figure Legend Snippet: Attachment and morphology of cultured human fungiform taste cells. ( a ) Primary cell cultures grown on collagen type-1-coated plates were imaged after 2 days. ( b ) Cells from human fungiform papillae grew for up to 2–4 weeks under attached cell clusters. ( c and d ) Represent day 2 and 4 weeks after harvesting, respectively. During this period we did not observe growth of cells with the appearance of non-taste epithelial cells.

    Techniques Used: Cell Culture

    39) Product Images from "Fluorescence Imaging of Interscapular Brown Adipose Tissue in Living Mice †"

    Article Title: Fluorescence Imaging of Interscapular Brown Adipose Tissue in Living Mice †

    Journal: Journal of materials chemistry. B, Materials for biology and medicine

    doi: 10.1039/C4TB01914H

    [A] Digestion of BAT with collagenase I produces an adipose fraction harboring brown adipocytes and a stromal fraction composed primarily of endothelium separated by a fluid portion. [B] Deep-red fluorescence intensity images of centrifugation tubes containing
    Figure Legend Snippet: [A] Digestion of BAT with collagenase I produces an adipose fraction harboring brown adipocytes and a stromal fraction composed primarily of endothelium separated by a fluid portion. [B] Deep-red fluorescence intensity images of centrifugation tubes containing

    Techniques Used: Fluorescence, Centrifugation

    40) Product Images from "Wntless regulates lipogenic gene expression in adipocytes and protects against diet-induced metabolic dysfunction"

    Article Title: Wntless regulates lipogenic gene expression in adipocytes and protects against diet-induced metabolic dysfunction

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2020.100992

    Canonical Wnt signaling is active in cultured adipocytes and Wntless is upregulated by diet-induced obesity . ( A–B ) MSCs isolated from C57BL/6J mice were cultured under standard conditions and induced to differentiate. Wntless gene (n = 6) and protein expression at indicated days of differentiation. ( C ) Wntless gene expression in the stromal-vascular (SVF) and adipocyte (Ads) fractions isolated from epididymal (eWAT) and inguinal (iWAT) white adipose tissues of C57BL/6J mice (males; n = 6). ( D ) Representative immunoblot of Wntless protein expression across C57BL/6J mouse tissues (asWAT, anterior subcutaneous WAT; pWAT, perirenal WAT; and BAT, brown adipose tissue); adiponectin, laminin, and Ponceau S were included as controls. ( E–F ) Regulation of Wntless gene and protein expression in eWAT and iWAT of 16-week-old wild-type mice fed a normal chow diet (NCD) or 8 weeks of high-fat diet (HFD) (males; n = 6). ( G ) Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 adipocytes vs day 0 cultured MSCs (n = 4). ( H ) Select Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 cultured adipocytes treated with recombinant Wnt3a (20 ng/ml) for 4 h (n = 4). RNA expression normalized to PPIA. Data are presented as mean ± S.D. ∗ indicates significance at p
    Figure Legend Snippet: Canonical Wnt signaling is active in cultured adipocytes and Wntless is upregulated by diet-induced obesity . ( A–B ) MSCs isolated from C57BL/6J mice were cultured under standard conditions and induced to differentiate. Wntless gene (n = 6) and protein expression at indicated days of differentiation. ( C ) Wntless gene expression in the stromal-vascular (SVF) and adipocyte (Ads) fractions isolated from epididymal (eWAT) and inguinal (iWAT) white adipose tissues of C57BL/6J mice (males; n = 6). ( D ) Representative immunoblot of Wntless protein expression across C57BL/6J mouse tissues (asWAT, anterior subcutaneous WAT; pWAT, perirenal WAT; and BAT, brown adipose tissue); adiponectin, laminin, and Ponceau S were included as controls. ( E–F ) Regulation of Wntless gene and protein expression in eWAT and iWAT of 16-week-old wild-type mice fed a normal chow diet (NCD) or 8 weeks of high-fat diet (HFD) (males; n = 6). ( G ) Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 adipocytes vs day 0 cultured MSCs (n = 4). ( H ) Select Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 cultured adipocytes treated with recombinant Wnt3a (20 ng/ml) for 4 h (n = 4). RNA expression normalized to PPIA. Data are presented as mean ± S.D. ∗ indicates significance at p

    Techniques Used: Cell Culture, Isolation, Mouse Assay, Expressing, RNA Sequencing Assay, Recombinant, RNA Expression

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    Isolation:

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    Incubation:

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    Mouse Assay:

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    Worthington Biochemical collagenase 1 mg ml worthington biochemical co nj usa
    Collagenase 1 Mg Ml Worthington Biochemical Co Nj Usa, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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