enzyme-linked immunosorbent assay elisa kit Search Results


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  • 96
    Thermo Fisher elisa kits
    Elisa Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 8254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore elisa kit
    Elisa Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2068 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher elisa kit
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    Thermo Fisher elisa
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    Becton Dickinson elisa kits
    The effects of ESAT-6 on DC cytokine production are not mimicked by Ag85A and are not due to contaminants in recombinant ESAT-6 or cell death A. iDCs were matured by stimulation with LPS/CD40LT, with or without ESAT-6 or Ag85A at different concentrations as indicated. Twenty-four h later, culture supernatants were collected and <t>IL-12p70,</t> IL-23 and IL-1β levels were measured by <t>ELISA.</t> B. iDCs were stimulated with LPS/CD40LT, in the presence of ESAT-6 preparations, depleted or sham-depleted of ESAT-6 with a nickel column, or with a depletion buffer without ESAT-6. Twenty four h later, supernatants were harvested and IL-12 levels were measured. C. iDCs were stimulated with LPS/CD40LT, with or without ESAT-6, for 24 h, and cell viability was evaluated with a MTT assay. For all panels, means and SEMs are shown. *, p
    Elisa Kits, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 3147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam elisa kit
    The effects of ESAT-6 on DC cytokine production are not mimicked by Ag85A and are not due to contaminants in recombinant ESAT-6 or cell death A. iDCs were matured by stimulation with LPS/CD40LT, with or without ESAT-6 or Ag85A at different concentrations as indicated. Twenty-four h later, culture supernatants were collected and <t>IL-12p70,</t> IL-23 and IL-1β levels were measured by <t>ELISA.</t> B. iDCs were stimulated with LPS/CD40LT, in the presence of ESAT-6 preparations, depleted or sham-depleted of ESAT-6 with a nickel column, or with a depletion buffer without ESAT-6. Twenty four h later, supernatants were harvested and IL-12 levels were measured. C. iDCs were stimulated with LPS/CD40LT, with or without ESAT-6, for 24 h, and cell viability was evaluated with a MTT assay. For all panels, means and SEMs are shown. *, p
    Elisa Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 1611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical elisa kit
    HTLV-1-induced <t>LTB4</t> secretion depends on Tax-induced cPLA2γ overexpression. ( a ) Tax-expressing Jurkat cells secrete significant levels of LTB4. Jurkat cells were transduced with lentiviral vectors encoding GFP alone or together with the viral regulatory proteins Tax and HBZ. Protein expression was detected by western blot 48 h post transduction (right panel). Spontaneous LTB4 secretion levels were determined by <t>ELISA.</t> P value
    Elisa Kit, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 94/100, based on 1503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher enzyme linked immunosorbent assay elisa kits
    HTLV-1-induced <t>LTB4</t> secretion depends on Tax-induced cPLA2γ overexpression. ( a ) Tax-expressing Jurkat cells secrete significant levels of LTB4. Jurkat cells were transduced with lentiviral vectors encoding GFP alone or together with the viral regulatory proteins Tax and HBZ. Protein expression was detected by western blot 48 h post transduction (right panel). Spontaneous LTB4 secretion levels were determined by <t>ELISA.</t> P value
    Enzyme Linked Immunosorbent Assay Elisa Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1715 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam elisa kits
    Koumine (KM) enhances LPS-induced astrocyte autophagy and attenuates astrocyte activation, as well as inflammation response in LPS-exposed primary astrocytes. Cultured astrocytes were exposed to koumine (0, 25, 50, or 100 μM) for 12 h prior to exposure to LPS for an additional 24 h before testing. Astrocytes in the control group were incubated with standard culture medium lacking koumine and LPS. Western blot (A) and densitometric quantification of GFAP (B) , Beclin 1 (C) , LC3-I/LC3-II (D) , P62 (E) , and GAPDH were performed in LPS-exposed astrocytes. <t>Elisa</t> were performed to explore the LPS-induced production of <t>TNF-α</t> (F) and IL-1β (G) in cultured astrocytes. Data are presented as the mean ± SEM. of three independent experiments. ### P
    Elisa Kits, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 1066 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Crystal Chem Inc ultra sensitive mouse insulin elisa kit
    Koumine (KM) enhances LPS-induced astrocyte autophagy and attenuates astrocyte activation, as well as inflammation response in LPS-exposed primary astrocytes. Cultured astrocytes were exposed to koumine (0, 25, 50, or 100 μM) for 12 h prior to exposure to LPS for an additional 24 h before testing. Astrocytes in the control group were incubated with standard culture medium lacking koumine and LPS. Western blot (A) and densitometric quantification of GFAP (B) , Beclin 1 (C) , LC3-I/LC3-II (D) , P62 (E) , and GAPDH were performed in LPS-exposed astrocytes. <t>Elisa</t> were performed to explore the LPS-induced production of <t>TNF-α</t> (F) and IL-1β (G) in cultured astrocytes. Data are presented as the mean ± SEM. of three independent experiments. ### P
    Ultra Sensitive Mouse Insulin Elisa Kit, supplied by Crystal Chem Inc, used in various techniques. Bioz Stars score: 93/100, based on 1190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical eia kit
    Letrozole reduced <t>17β-estradiol</t> levels in the brain and shortened seizure latency. Mice were fed a diet containing cottonseed oil or cottonseed oil supplemented with DHA and injected with letrozole (10 mg/kg) intraperitoneally every other day for 28 days. ( a ) The 17β-estradiol levels in the cerebral cortex were determined by <t>EIA.</t> The values represent the mean ± S.E. (n = 7 animals in each group). Data were analyzed using Student’s t -test. ** P
    Eia Kit, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 94/100, based on 1107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem elisa kit
    <t>Ntn4</t> was overexpressed in GCs and negatively correlated with patient survival A. IHC staining of human GC tissues using Ntn4-specific antibody, as described in Methods; Magnification, × 200. B–C. Classification of samples according to the intensity of staining of Ntn4 expression ( n = 82, p = 0.001). D. <t>ELISA</t> analysis to determine serum level of Ntn4 in GC patients and normal controls ( n = 52, p
    Elisa Kit, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 841 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    USCN Life elisa kit
    <t>CFI</t> bioactivity in a cohort of subjects with staged AMD and non-AMD normals. Subjects with AMD were staged according to the AREDS classification [ 24 ]. CFI bioactivity was determined by measuring the conversion of C3b to iC3b using <t>ELISA</t> for iC3b. Plasma dilutions (0.01–25 μl) were exposed to 80 μg/ml of each CFH and C3b and incubated at 37°C for 2 hours. Levels of iC3b were measured using ELISA. Samples from the same individual were run in the same assay as singlets. To control for the multiple assays conducted, each assay included a control sample. Data were fitted to a 4-parameter logistic equation and EC 50 s estimated. These values were converted to bioactivity in U/ μ g of CFI (1 mU = 1 / EC 50 in pg/ml). The logarithm of the bioactivity for the experimental samples and control plasma samples were analyzed by a mixed-model ANCOVA, using the control sample as a covariate to correct for inter-run variability. Data are shown as the fitted means in the original scale ± SE estimated from the fitted values using the Taylor series expansion. N = 4 to 47 patients per group. Legends: AREDS, Age-Related Eye Disease Study stages I through III, GA, geographic atrophy, Wet-Inactive, wet active AMD.
    Elisa Kit, supplied by USCN Life, used in various techniques. Bioz Stars score: 94/100, based on 1303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ALPCO elisa kit
    EREG stimulates leptin secretion and lipolysis in mice with diet-induced obesity (DIO). (A) DIO WT male mice (n=7/ group) were injected with 100μL PBS (Veh) with and without EREG (1.5ng/g body weight or 20ng/per iAb depot) into both epididymal iAb fat pads every other day for 2 weeks. Total mRNA was isolated from one whole iAb fad pad. Expression of <t>Lep</t> was measured using NanoString assay. Normalized data represent mean±SD, n=5; Mann-Whitney U test. (B) Expression of LEP protein levels were measured in plasma in the same mice group by <t>ELISA.</t> Data (mean±SD, n=5/group). Independent Student’s t -test. (C) Non-esterified fatty acids (NEFA) and (D) TG concentrations in plasma are shown as mean ± SD, n=5. Independent Student’s t -test.
    Elisa Kit, supplied by ALPCO, used in various techniques. Bioz Stars score: 94/100, based on 904 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MyBiosource elisa kit
    Effects of the <t>β2-GPI</t> DNA vaccine and FK506 treatment on β2-GPI-specific Treg cell response. Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the percentage of Foxp3-expressing CD4+ T cells was determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± standard deviation (SD) of six mice from three independent experiments. (B) The culture supernatants were collected, and IL-10 and TGF-β production was analyzed in triplicate by <t>ELISA.</t> The data are presented as the means ± SD of triplicate assays from six mice/group. ns p > 0.05, **p
    Elisa Kit, supplied by MyBiosource, used in various techniques. Bioz Stars score: 94/100, based on 932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher enzyme linked immunosorbent assay kits
    Effects of the <t>β2-GPI</t> DNA vaccine and FK506 treatment on β2-GPI-specific Treg cell response. Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the percentage of Foxp3-expressing CD4+ T cells was determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± standard deviation (SD) of six mice from three independent experiments. (B) The culture supernatants were collected, and IL-10 and TGF-β production was analyzed in triplicate by <t>ELISA.</t> The data are presented as the means ± SD of triplicate assays from six mice/group. ns p > 0.05, **p
    Enzyme Linked Immunosorbent Assay Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cloud-Clone elisa kit
    Comparison of serum soluble TLR2 <t>(sTLR2)</t> levels in the discovery cohort, validation cohort, and pooled data measured by enzyme-linked immunosorbent assay and analysis of pooled data. The data are presented as mean ± SD. (A) sTLR2 measured in the serum samples from RRMS patients and HC in the discovery cohort ( n = 21 RRMS, 24 HC). Mann–Whitney test showed significantly higher sTLR2 values in the RRMS patients ( P
    Elisa Kit, supplied by Cloud-Clone, used in various techniques. Bioz Stars score: 94/100, based on 860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The construction of human ALIM infected by Streptococcus . A. The relative LDH vitality of supernates in ALIM at 4 h, 24 h and 48 h. B. The relative expression of IL-6, IL-8 and <t>TNF-α</t> of supernates in ALIM. LDH, lactate
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    The construction of human ALIM infected by Streptococcus . A. The relative LDH vitality of supernates in ALIM at 4 h, 24 h and 48 h. B. The relative expression of IL-6, IL-8 and <t>TNF-α</t> of supernates in ALIM. LDH, lactate
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    IDEXX elisa kit
    Evaluation of specificity and reactivity of six representative sdAbs against SIV-NP. Notes: ( A ) The <t>sdAb</t> of one representative member of each family extracted from IPTG-induced E. coli TG1 colonies was checked for their specific binding to the immobilized SIV-NP using an indirect <t>ELISA.</t> PEDV-N was used as an irrelevant protein control. ( B ) The reactivity of six representative sdAbs against SIV-NP was detected by indirect ELISA. Abbreviations: sdAb, single-domain antibody; IPTG, isopropyl-β-D-thiogalactopyranoside; SIV, swine influenza virus; NP, nucleoprotein; PEDV, porcine epidemic diarrhea virus; N, nucleocapsid.
    Elisa Kit, supplied by IDEXX, used in various techniques. Bioz Stars score: 94/100, based on 814 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher enzyme linked immunosorbent assay elisa kit
    Evaluation of specificity and reactivity of six representative sdAbs against SIV-NP. Notes: ( A ) The <t>sdAb</t> of one representative member of each family extracted from IPTG-induced E. coli TG1 colonies was checked for their specific binding to the immobilized SIV-NP using an indirect <t>ELISA.</t> PEDV-N was used as an irrelevant protein control. ( B ) The reactivity of six representative sdAbs against SIV-NP was detected by indirect ELISA. Abbreviations: sdAb, single-domain antibody; IPTG, isopropyl-β-D-thiogalactopyranoside; SIV, swine influenza virus; NP, nucleoprotein; PEDV, porcine epidemic diarrhea virus; N, nucleocapsid.
    Enzyme Linked Immunosorbent Assay Elisa Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher amyloid beta 42 human elisa kit
    Evaluation of specificity and reactivity of six representative sdAbs against SIV-NP. Notes: ( A ) The <t>sdAb</t> of one representative member of each family extracted from IPTG-induced E. coli TG1 colonies was checked for their specific binding to the immobilized SIV-NP using an indirect <t>ELISA.</t> PEDV-N was used as an irrelevant protein control. ( B ) The reactivity of six representative sdAbs against SIV-NP was detected by indirect ELISA. Abbreviations: sdAb, single-domain antibody; IPTG, isopropyl-β-D-thiogalactopyranoside; SIV, swine influenza virus; NP, nucleoprotein; PEDV, porcine epidemic diarrhea virus; N, nucleocapsid.
    Amyloid Beta 42 Human Elisa Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 793 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Crystal Chem Inc elisa kit
    EREG stimulates leptin secretion and lipolysis in mice with diet-induced obesity (DIO). (A) DIO WT male mice (n=7/ group) were injected with 100μL PBS (Veh) with and without EREG (1.5ng/g body weight or 20ng/per <t>iAb</t> depot) into both epididymal iAb fat pads every other day for 2 weeks. Total mRNA was isolated from one whole iAb fad pad. Expression of Lep was measured using NanoString assay. Normalized data represent mean±SD, n=5; Mann-Whitney U test. (B) Expression of LEP protein levels were measured in plasma in the same mice group by <t>ELISA.</t> Data (mean±SD, n=5/group). Independent Student’s t -test. (C) Non-esterified fatty acids (NEFA) and (D) TG concentrations in plasma are shown as mean ± SD, n=5. Independent Student’s t -test.
    Elisa Kit, supplied by Crystal Chem Inc, used in various techniques. Bioz Stars score: 93/100, based on 585 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tuft cells require Dclk1 expression to mediate its paracrine function. ( a ) Western blot analysis was performed to identify the protein expression level of COX2 in the vector control and DCLK1-overexpressing YAMC cells. ( b ) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to COX2. Lower bands represent IgG heavy and short chains. ( c ) Co-immunoprecipitation: IEC protein extracts from Dclk1 f/f mice at homeostasis and 24 h post-TBI were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to Cox2. Lower bands represent IgG heavy and short chains. ( d ) <t>PGE2</t> <t>EIA</t> assay: Cell culture spent media collected and utilized for analyzing PGE2 levels. ( e ) Bar graph represents the percent colony formation of YAMC cells of vector control, Dclk1-overexpressing, PGE2-treated, and co-culture (YAMC + YAMC Dclk1-overexpression) cells at baseline and 48 h post-radiation. ( f ) Proposed Mechanism: Tuft cells regulates ATM mediated DDR in response to injury to enhance intestinal epithelial survival and self-renewal for effective restitution and function. In response to radiation-induced injury, Dclk1 + tuft cells enhance COX2 signaling for the paracrine regulation of IECs/ISCs. Dclk1 expression is critical for ATM activation for the DNA DSB repair response. ATM activation significantly controls DNA repair, apoptosis, and the cell cycle. Cell survival and self-renewal depends upon cellular DNA integrity. Dclk1 regulation of the ATM pathway enhances cell survival and self-renewal for effective intestinal regeneration after TBI.
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    Image Search Results


    The effects of ESAT-6 on DC cytokine production are not mimicked by Ag85A and are not due to contaminants in recombinant ESAT-6 or cell death A. iDCs were matured by stimulation with LPS/CD40LT, with or without ESAT-6 or Ag85A at different concentrations as indicated. Twenty-four h later, culture supernatants were collected and IL-12p70, IL-23 and IL-1β levels were measured by ELISA. B. iDCs were stimulated with LPS/CD40LT, in the presence of ESAT-6 preparations, depleted or sham-depleted of ESAT-6 with a nickel column, or with a depletion buffer without ESAT-6. Twenty four h later, supernatants were harvested and IL-12 levels were measured. C. iDCs were stimulated with LPS/CD40LT, with or without ESAT-6, for 24 h, and cell viability was evaluated with a MTT assay. For all panels, means and SEMs are shown. *, p

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

    Article Title: Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1

    doi: 10.4049/jimmunol.1200573

    Figure Lengend Snippet: The effects of ESAT-6 on DC cytokine production are not mimicked by Ag85A and are not due to contaminants in recombinant ESAT-6 or cell death A. iDCs were matured by stimulation with LPS/CD40LT, with or without ESAT-6 or Ag85A at different concentrations as indicated. Twenty-four h later, culture supernatants were collected and IL-12p70, IL-23 and IL-1β levels were measured by ELISA. B. iDCs were stimulated with LPS/CD40LT, in the presence of ESAT-6 preparations, depleted or sham-depleted of ESAT-6 with a nickel column, or with a depletion buffer without ESAT-6. Twenty four h later, supernatants were harvested and IL-12 levels were measured. C. iDCs were stimulated with LPS/CD40LT, with or without ESAT-6, for 24 h, and cell viability was evaluated with a MTT assay. For all panels, means and SEMs are shown. *, p

    Article Snippet: To generate immature DCs, CD14+ cells were cultured at 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with 10% heat-inactivated pooled human serum (Atlanta Biologicals), 100 units or 100 µg/ml penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids (all from Invitrogen), and 25 ng/ml human granulocyte-macrophage-colony-stimulating factor and IL-4 (both from R & D systems) for 3–5 d. The immature DCs were further stimulated with human CD40LT (Immunex, Seattle, Washington) at 2.5 µg/ml, plus LPS (Sigma) at 1 µg/ml, with or without ESAT-6, for 24 h. In some experiments, CD3+ cells were cultured at 2 × 106 /ml with autologous or allogeneic DCs in 96-well flat-bottom plates for 3–5 d. Cell-free supernatants were collected and cytokine concentrations were measured by ELISA, using capture and detection antibodies for IFN-γ and TNF-α (BD Biosciences), and ELISA kits for IL-2, IL-12p70 (both from BD Biosciences), IL-17, IL-8 (both from R & D systems), IL-1β, IL-23 (both from Mabtech), and IL-6 (BioLegend).

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Nickel Column, MTT Assay

    ESAT-6-treated DCs stimulate T cell IL-17 production through IL-23 and IL-1β Purified CD3 + cells from healthy donors were stimulated with plate-bound anti-CD3 plus anti-CD28 in a 96-well plate, with each well containing 100 µl of RPMI-1640 and 10% human serum and 100 µl of conditioned medium from DCs, matured under the conditions as indicated. Forty-eight h later, supernatants were harvested and IL-17 ( A ) and IFN-γ ( B ) levels were measured by ELISA. C , Purified CD3 + cells were cultured in 180 µl of RPMI-1640 with 10% human serum and 20 µl of conditioned medium from DCs, matured by LPS/CD40LT in the presence of ESAT-6. Some cells were treated with neutralizing Abs or isotype control IgG for 1 h, prior to stimulation with anti-CD3 plus anti-CD28 in a 96-well plate. Forty-eight h later, supernatants were harvested and IL-17 levels were measured by ELISA. Means and SEMs are shown for all three panels. *, p

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

    Article Title: Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1

    doi: 10.4049/jimmunol.1200573

    Figure Lengend Snippet: ESAT-6-treated DCs stimulate T cell IL-17 production through IL-23 and IL-1β Purified CD3 + cells from healthy donors were stimulated with plate-bound anti-CD3 plus anti-CD28 in a 96-well plate, with each well containing 100 µl of RPMI-1640 and 10% human serum and 100 µl of conditioned medium from DCs, matured under the conditions as indicated. Forty-eight h later, supernatants were harvested and IL-17 ( A ) and IFN-γ ( B ) levels were measured by ELISA. C , Purified CD3 + cells were cultured in 180 µl of RPMI-1640 with 10% human serum and 20 µl of conditioned medium from DCs, matured by LPS/CD40LT in the presence of ESAT-6. Some cells were treated with neutralizing Abs or isotype control IgG for 1 h, prior to stimulation with anti-CD3 plus anti-CD28 in a 96-well plate. Forty-eight h later, supernatants were harvested and IL-17 levels were measured by ELISA. Means and SEMs are shown for all three panels. *, p

    Article Snippet: To generate immature DCs, CD14+ cells were cultured at 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with 10% heat-inactivated pooled human serum (Atlanta Biologicals), 100 units or 100 µg/ml penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids (all from Invitrogen), and 25 ng/ml human granulocyte-macrophage-colony-stimulating factor and IL-4 (both from R & D systems) for 3–5 d. The immature DCs were further stimulated with human CD40LT (Immunex, Seattle, Washington) at 2.5 µg/ml, plus LPS (Sigma) at 1 µg/ml, with or without ESAT-6, for 24 h. In some experiments, CD3+ cells were cultured at 2 × 106 /ml with autologous or allogeneic DCs in 96-well flat-bottom plates for 3–5 d. Cell-free supernatants were collected and cytokine concentrations were measured by ELISA, using capture and detection antibodies for IFN-γ and TNF-α (BD Biosciences), and ELISA kits for IL-2, IL-12p70 (both from BD Biosciences), IL-17, IL-8 (both from R & D systems), IL-1β, IL-23 (both from Mabtech), and IL-6 (BioLegend).

    Techniques: Purification, Enzyme-linked Immunosorbent Assay, Cell Culture

    DC matured in the presence of ESAT-6 inhibits production of Th1 cytokines and enhances production of IL-17 by allogeneic T cells iDCs generated from four healthy donors were matured with LPS/CD40LT, with or without 20 µg/ml of ESAT-6. After extensive washing to remove free ESAT-6 and LPS/CD40LT, varying number of mDC ranging from 4 × 10 3 to 4 × 10 4 were incubated with 4 × 10 5 CFSE-labeled, allogeneic CD4 + T cells at indicated ratios. A. After 4 days, the percentages of proliferating cells were determined by flow cytometry. A representative result is shown. B. The means and SEMs of the percentage of proliferated CD4 + cells are shown from four experiments. C–E. The supernatants from cocultures of mDCs and allogeneic CD4 + T cells from five donors were collected after 24 h for measurement of IL-2 ( D ) and after 96 h for measurement of IFN-γ ( C ) and IL-17 ( E ) by ELISA. Means and SEMs are shown. *, p

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

    Article Title: Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1

    doi: 10.4049/jimmunol.1200573

    Figure Lengend Snippet: DC matured in the presence of ESAT-6 inhibits production of Th1 cytokines and enhances production of IL-17 by allogeneic T cells iDCs generated from four healthy donors were matured with LPS/CD40LT, with or without 20 µg/ml of ESAT-6. After extensive washing to remove free ESAT-6 and LPS/CD40LT, varying number of mDC ranging from 4 × 10 3 to 4 × 10 4 were incubated with 4 × 10 5 CFSE-labeled, allogeneic CD4 + T cells at indicated ratios. A. After 4 days, the percentages of proliferating cells were determined by flow cytometry. A representative result is shown. B. The means and SEMs of the percentage of proliferated CD4 + cells are shown from four experiments. C–E. The supernatants from cocultures of mDCs and allogeneic CD4 + T cells from five donors were collected after 24 h for measurement of IL-2 ( D ) and after 96 h for measurement of IFN-γ ( C ) and IL-17 ( E ) by ELISA. Means and SEMs are shown. *, p

    Article Snippet: To generate immature DCs, CD14+ cells were cultured at 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with 10% heat-inactivated pooled human serum (Atlanta Biologicals), 100 units or 100 µg/ml penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids (all from Invitrogen), and 25 ng/ml human granulocyte-macrophage-colony-stimulating factor and IL-4 (both from R & D systems) for 3–5 d. The immature DCs were further stimulated with human CD40LT (Immunex, Seattle, Washington) at 2.5 µg/ml, plus LPS (Sigma) at 1 µg/ml, with or without ESAT-6, for 24 h. In some experiments, CD3+ cells were cultured at 2 × 106 /ml with autologous or allogeneic DCs in 96-well flat-bottom plates for 3–5 d. Cell-free supernatants were collected and cytokine concentrations were measured by ELISA, using capture and detection antibodies for IFN-γ and TNF-α (BD Biosciences), and ELISA kits for IL-2, IL-12p70 (both from BD Biosciences), IL-17, IL-8 (both from R & D systems), IL-1β, IL-23 (both from Mabtech), and IL-6 (BioLegend).

    Techniques: Generated, Incubation, Labeling, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    DCs matured with ESAT-6 stimulate IL-17 and inhibit IFN-γ production by M. tuberculosis -responsive T cells iDCs from four donors with latent tuberculosis infection were matured with LPS/CD40LT, with or without ESAT-6, for 24 h. The cells were then washed and incubated with autologous CD3 + cells at different ratios, and stimulated with heat-killed Mtb at 2.5 µg/ml (left panels) or infected with H37Rv at a MOI of 20 (right panels). Forty-eight hrs later, the supernatants were harvested and IFN-γ and IL-17 levels were measured by ELISA. Means and SEMs are shown. *, p

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

    Article Title: Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1

    doi: 10.4049/jimmunol.1200573

    Figure Lengend Snippet: DCs matured with ESAT-6 stimulate IL-17 and inhibit IFN-γ production by M. tuberculosis -responsive T cells iDCs from four donors with latent tuberculosis infection were matured with LPS/CD40LT, with or without ESAT-6, for 24 h. The cells were then washed and incubated with autologous CD3 + cells at different ratios, and stimulated with heat-killed Mtb at 2.5 µg/ml (left panels) or infected with H37Rv at a MOI of 20 (right panels). Forty-eight hrs later, the supernatants were harvested and IFN-γ and IL-17 levels were measured by ELISA. Means and SEMs are shown. *, p

    Article Snippet: To generate immature DCs, CD14+ cells were cultured at 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with 10% heat-inactivated pooled human serum (Atlanta Biologicals), 100 units or 100 µg/ml penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids (all from Invitrogen), and 25 ng/ml human granulocyte-macrophage-colony-stimulating factor and IL-4 (both from R & D systems) for 3–5 d. The immature DCs were further stimulated with human CD40LT (Immunex, Seattle, Washington) at 2.5 µg/ml, plus LPS (Sigma) at 1 µg/ml, with or without ESAT-6, for 24 h. In some experiments, CD3+ cells were cultured at 2 × 106 /ml with autologous or allogeneic DCs in 96-well flat-bottom plates for 3–5 d. Cell-free supernatants were collected and cytokine concentrations were measured by ELISA, using capture and detection antibodies for IFN-γ and TNF-α (BD Biosciences), and ELISA kits for IL-2, IL-12p70 (both from BD Biosciences), IL-17, IL-8 (both from R & D systems), IL-1β, IL-23 (both from Mabtech), and IL-6 (BioLegend).

    Techniques: Infection, Incubation, Enzyme-linked Immunosorbent Assay

    DCs matured in the presence of ESAT-6 produce less IL-12 and more IL-23 and IL-1β iDCs were stimulated with LPS/CD40LT, with or without ESAT-6, and some cells were incubated with ESAT-6 only. Twenty-four hrs post stimulation, the supernatants were collected and levels of IL-12p70 ( A ), IL-23 ( B ), IL-1β ( C ), TNFα ( D ), IL-6 ( E ) and IL-8 ( F ) were measured by ELISA. G . Some cells were treated with a caspase 1 inhibitor (20 µM) prior to stimulation with LPS/CD40LT and ESAT-6. Twenty-four h later, supernatants were collected and IL-1β levels were measured. The far right grey bar represents cells treated with DMSO only as a vehicle control. For all panels, means and SEMs are shown. *, p

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

    Article Title: Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1

    doi: 10.4049/jimmunol.1200573

    Figure Lengend Snippet: DCs matured in the presence of ESAT-6 produce less IL-12 and more IL-23 and IL-1β iDCs were stimulated with LPS/CD40LT, with or without ESAT-6, and some cells were incubated with ESAT-6 only. Twenty-four hrs post stimulation, the supernatants were collected and levels of IL-12p70 ( A ), IL-23 ( B ), IL-1β ( C ), TNFα ( D ), IL-6 ( E ) and IL-8 ( F ) were measured by ELISA. G . Some cells were treated with a caspase 1 inhibitor (20 µM) prior to stimulation with LPS/CD40LT and ESAT-6. Twenty-four h later, supernatants were collected and IL-1β levels were measured. The far right grey bar represents cells treated with DMSO only as a vehicle control. For all panels, means and SEMs are shown. *, p

    Article Snippet: To generate immature DCs, CD14+ cells were cultured at 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with 10% heat-inactivated pooled human serum (Atlanta Biologicals), 100 units or 100 µg/ml penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids (all from Invitrogen), and 25 ng/ml human granulocyte-macrophage-colony-stimulating factor and IL-4 (both from R & D systems) for 3–5 d. The immature DCs were further stimulated with human CD40LT (Immunex, Seattle, Washington) at 2.5 µg/ml, plus LPS (Sigma) at 1 µg/ml, with or without ESAT-6, for 24 h. In some experiments, CD3+ cells were cultured at 2 × 106 /ml with autologous or allogeneic DCs in 96-well flat-bottom plates for 3–5 d. Cell-free supernatants were collected and cytokine concentrations were measured by ELISA, using capture and detection antibodies for IFN-γ and TNF-α (BD Biosciences), and ELISA kits for IL-2, IL-12p70 (both from BD Biosciences), IL-17, IL-8 (both from R & D systems), IL-1β, IL-23 (both from Mabtech), and IL-6 (BioLegend).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    Differential effects of M. tuberculosis H37Rv and its esat-6 deletion mutant on DC cytokine production iDCs from four donors were infected with live H37Rv and its esat-6 deletion mutant, H37RvΔ3875, at a multiplicity of infection of 10. Four h later, the cells were stimulated with LPS plus CD40LT for 24 h, culture supernatants were collected and levels of IL-12p70 (A), IL-23 (B), and IL-1β (C) were measured by ELISA. Means and SEMs of results from four donors are shown. *, p

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

    Article Title: Early Secreted Antigenic Target of 6- kD Protein of Mycobacterium tuberculosis Primes Dendritic Cells to Stimulate Th17 and Inhibit Th1 Immune Responses 1

    doi: 10.4049/jimmunol.1200573

    Figure Lengend Snippet: Differential effects of M. tuberculosis H37Rv and its esat-6 deletion mutant on DC cytokine production iDCs from four donors were infected with live H37Rv and its esat-6 deletion mutant, H37RvΔ3875, at a multiplicity of infection of 10. Four h later, the cells were stimulated with LPS plus CD40LT for 24 h, culture supernatants were collected and levels of IL-12p70 (A), IL-23 (B), and IL-1β (C) were measured by ELISA. Means and SEMs of results from four donors are shown. *, p

    Article Snippet: To generate immature DCs, CD14+ cells were cultured at 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with 10% heat-inactivated pooled human serum (Atlanta Biologicals), 100 units or 100 µg/ml penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids (all from Invitrogen), and 25 ng/ml human granulocyte-macrophage-colony-stimulating factor and IL-4 (both from R & D systems) for 3–5 d. The immature DCs were further stimulated with human CD40LT (Immunex, Seattle, Washington) at 2.5 µg/ml, plus LPS (Sigma) at 1 µg/ml, with or without ESAT-6, for 24 h. In some experiments, CD3+ cells were cultured at 2 × 106 /ml with autologous or allogeneic DCs in 96-well flat-bottom plates for 3–5 d. Cell-free supernatants were collected and cytokine concentrations were measured by ELISA, using capture and detection antibodies for IFN-γ and TNF-α (BD Biosciences), and ELISA kits for IL-2, IL-12p70 (both from BD Biosciences), IL-17, IL-8 (both from R & D systems), IL-1β, IL-23 (both from Mabtech), and IL-6 (BioLegend).

    Techniques: Mutagenesis, Infection, Enzyme-linked Immunosorbent Assay

    EV71 proteins was participated in the activation of NLRP3 inflammasome. ( A and B ) TPA-differentiated THP-1 cells were mock-infected for 2 h (lanes 1–3) or infection with EV71 (MOI = 20) for 2 h (lanes 7–9), treated with 100 μM of cycloheximide (CHX) for 1 h, washed with PBS, and then grown for additional 22 h. TPA-differentiated THP-1 cells were treated with 100 μM of cycloheximide (CHX) for 1 h, washed with PBS, grown for additional 20 h, and then treated with 2 μM Nigericin for 2 h as a positive control (lanes 4–6). Supernatants of the cell cultures were analyzed by ELISA for IL-1β secretion (A). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-Casp-1, NLRP3, EV71 3D, and GAPDH proteins (Lys) (B). ( C ) HEK293T cells were transfected with plasmids encoding pro-IL-1β, with(+) or without(-) Flag-pro-caspsase-1, with(+) or without(-) Flag-NLRP3 and with(+) or without(-) Flag-ASC to established NLRP3 inflammasome. Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, NLRP3, ASC, and GAPDH (Lys). ( D and E ) HEK293T cells were transfected with plasmids encoding pro-IL-1β, Flag-pro-Casp-1, Flag-NLRP3, Flag-ASC, and EV71 non-structure proteins (2A, 2B, 2C, 3A, 3C, and 3D). Using the GFP as a negative control. Supernatants were analyzed by ELISA for IL-1β secretion (D). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-caspase-1, NLRP3, GFP and GAPDH (E). ( F and G ) TPA-differentiated THP-1 cells was infected by the EV71 by a time dependent (MOI = 20) for 3, 6, 12, 24, and 36 h. Supernatants were analyzed by ELISA for IL-1β secretion (F). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, NLRP3, EIF4G, EV71 3C, EC71 3D, and GAPDH proteins (Lys) (G). ( H ) RD cells was infected with EV71 at MOI = 1 for 2, 4, 6, 8, and 12 h. Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for EIF4G, EV71 3C, EC71 3D, and GAPDH proteins (H). Data shown are means ± SEM, *p

    Journal: PLoS Pathogens

    Article Title: EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex

    doi: 10.1371/journal.ppat.1006123

    Figure Lengend Snippet: EV71 proteins was participated in the activation of NLRP3 inflammasome. ( A and B ) TPA-differentiated THP-1 cells were mock-infected for 2 h (lanes 1–3) or infection with EV71 (MOI = 20) for 2 h (lanes 7–9), treated with 100 μM of cycloheximide (CHX) for 1 h, washed with PBS, and then grown for additional 22 h. TPA-differentiated THP-1 cells were treated with 100 μM of cycloheximide (CHX) for 1 h, washed with PBS, grown for additional 20 h, and then treated with 2 μM Nigericin for 2 h as a positive control (lanes 4–6). Supernatants of the cell cultures were analyzed by ELISA for IL-1β secretion (A). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-Casp-1, NLRP3, EV71 3D, and GAPDH proteins (Lys) (B). ( C ) HEK293T cells were transfected with plasmids encoding pro-IL-1β, with(+) or without(-) Flag-pro-caspsase-1, with(+) or without(-) Flag-NLRP3 and with(+) or without(-) Flag-ASC to established NLRP3 inflammasome. Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, NLRP3, ASC, and GAPDH (Lys). ( D and E ) HEK293T cells were transfected with plasmids encoding pro-IL-1β, Flag-pro-Casp-1, Flag-NLRP3, Flag-ASC, and EV71 non-structure proteins (2A, 2B, 2C, 3A, 3C, and 3D). Using the GFP as a negative control. Supernatants were analyzed by ELISA for IL-1β secretion (D). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-caspase-1, NLRP3, GFP and GAPDH (E). ( F and G ) TPA-differentiated THP-1 cells was infected by the EV71 by a time dependent (MOI = 20) for 3, 6, 12, 24, and 36 h. Supernatants were analyzed by ELISA for IL-1β secretion (F). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, NLRP3, EIF4G, EV71 3C, EC71 3D, and GAPDH proteins (Lys) (G). ( H ) RD cells was infected with EV71 at MOI = 1 for 2, 4, 6, 8, and 12 h. Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for EIF4G, EV71 3C, EC71 3D, and GAPDH proteins (H). Data shown are means ± SEM, *p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) The concentrations of IL-1β in culture supernatants were measured by ELISA kit (BD Biosciences, San Jose, CA).

    Techniques: Activation Assay, Infection, Positive Control, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control

    EV71 3D facilitates the activation of NLRP3 inflammasome. ( A and B ) HEK293T cells were transfected with plasmids encoding pro-IL-1βFlag-pro-caspsase-1, with(+) or without(-) Flag-NLRP3, with(+) or without(-) Flag-ASC and with(+) or without(-) EV71 3D protein. Using the GFP as a negative control. Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, GFP, and GAPDH (A). Supernatants of the cell cultures were analyzed by ELISA for the detection of IL-1β secretion (B). ( C ) THP-1 cells were infected by control (CT) or 3D-encoding lentivirus. And then THP-1 cells were differentiated into macrophages with TPA. The mature (p17) form of IL-1β in the supernatants were analyzed by immunoblot (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, EV71 3D protein, and GAPDH (Lys). ( D ) ASC oligomerization in TPA-differentiated THP-1 cells which were infected by control (CT) or 3D-encoding lentivirus. TPA-differentiated THP-1 cells were treated with the 2 μM Nigericin for 2 h as a positive control. TPA-differentiated THP-1 cells were treated with DMSO as a negative control. ( E and F ) TPA-differentiated THP-1 cells which were infected by control (CT) or 3D-encoding lentivirus was treated with 2 μM Nigericin for 2 h, 5 mM ATP for 2 h or infection with Sendaivirus (MOI = 1) for 24 h. Supernatants were analyzed by ELISA for IL-1β secretion (E). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-caspase-1, EV71 3D protein, and GAPDH (Lys) (F). ( G and H ) TPA-differentiated THP-1 cells were infected with control (CT) or 3D-encoding lentivirus, treated with DMSO or 80 μM aurintricarboxylic acid (ATA) for 24 h, and treated with 5 mM ATP for 2 h. Supernatants were analyzed by ELISA for IL-1β secretion (G). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, EV71 3D, and GAPDH proteins (Lys) (H). ( I and J ) TPA-differentiated THP-1 cells were infected with EV71 at MOI = 20 for 2 h, treated with DMSO or 80 μM aurintricarboxylic acid (ATA), and the treated with 5 mM ATP for 2 h. At 24 h post-infection of EV71, supernatants were analyzed by ELISA for IL-1β secretion (I). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, EV71 3D, and GAPDH proteins (Lys) (J). Data shown are means ± SEM, *p

    Journal: PLoS Pathogens

    Article Title: EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex

    doi: 10.1371/journal.ppat.1006123

    Figure Lengend Snippet: EV71 3D facilitates the activation of NLRP3 inflammasome. ( A and B ) HEK293T cells were transfected with plasmids encoding pro-IL-1βFlag-pro-caspsase-1, with(+) or without(-) Flag-NLRP3, with(+) or without(-) Flag-ASC and with(+) or without(-) EV71 3D protein. Using the GFP as a negative control. Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, GFP, and GAPDH (A). Supernatants of the cell cultures were analyzed by ELISA for the detection of IL-1β secretion (B). ( C ) THP-1 cells were infected by control (CT) or 3D-encoding lentivirus. And then THP-1 cells were differentiated into macrophages with TPA. The mature (p17) form of IL-1β in the supernatants were analyzed by immunoblot (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for IL-1β, Caspase-1, EV71 3D protein, and GAPDH (Lys). ( D ) ASC oligomerization in TPA-differentiated THP-1 cells which were infected by control (CT) or 3D-encoding lentivirus. TPA-differentiated THP-1 cells were treated with the 2 μM Nigericin for 2 h as a positive control. TPA-differentiated THP-1 cells were treated with DMSO as a negative control. ( E and F ) TPA-differentiated THP-1 cells which were infected by control (CT) or 3D-encoding lentivirus was treated with 2 μM Nigericin for 2 h, 5 mM ATP for 2 h or infection with Sendaivirus (MOI = 1) for 24 h. Supernatants were analyzed by ELISA for IL-1β secretion (E). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-caspase-1, EV71 3D protein, and GAPDH (Lys) (F). ( G and H ) TPA-differentiated THP-1 cells were infected with control (CT) or 3D-encoding lentivirus, treated with DMSO or 80 μM aurintricarboxylic acid (ATA) for 24 h, and treated with 5 mM ATP for 2 h. Supernatants were analyzed by ELISA for IL-1β secretion (G). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, EV71 3D, and GAPDH proteins (Lys) (H). ( I and J ) TPA-differentiated THP-1 cells were infected with EV71 at MOI = 20 for 2 h, treated with DMSO or 80 μM aurintricarboxylic acid (ATA), and the treated with 5 mM ATP for 2 h. At 24 h post-infection of EV71, supernatants were analyzed by ELISA for IL-1β secretion (I). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, EV71 3D, and GAPDH proteins (Lys) (J). Data shown are means ± SEM, *p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) The concentrations of IL-1β in culture supernatants were measured by ELISA kit (BD Biosciences, San Jose, CA).

    Techniques: Activation Assay, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

    The production and secretion of IL-1β are induced by EV71 in macrophages. (A , B and C) TPA-differentiated THP-1 cells were infected by the EV71 virus by a time dependent (MOI = 20) for 24 h and 48 h. Supernatants were analyzed by ELISA for IL-1β secretion (A). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 were analyzed in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-caspase-1, EV71 3D, and GAPDH (B). The mRNA levels for the gene pro-IL-1β were quantified by real-time PCR (C). ( D , E and F ) TPA-differentiated THP-1 cells were infected by the EV71 virus by a dose dependent (MOI = 5, 10, 20) for 24 h. Using the LPS (1 μg/ml) for 6 h and 2 μM Nigericin for 30 min as a positive control. Supernatants were analyzed by ELISA for IL-1β secretion (D). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-Caspase-1, and EV71 3D protein and GAPDH (Lys) (E). The mRNA levels for the gene pro-IL-1β were quantified by real-time PCR (F). Data shown are means ± SEM, *p

    Journal: PLoS Pathogens

    Article Title: EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex

    doi: 10.1371/journal.ppat.1006123

    Figure Lengend Snippet: The production and secretion of IL-1β are induced by EV71 in macrophages. (A , B and C) TPA-differentiated THP-1 cells were infected by the EV71 virus by a time dependent (MOI = 20) for 24 h and 48 h. Supernatants were analyzed by ELISA for IL-1β secretion (A). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 were analyzed in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-caspase-1, EV71 3D, and GAPDH (B). The mRNA levels for the gene pro-IL-1β were quantified by real-time PCR (C). ( D , E and F ) TPA-differentiated THP-1 cells were infected by the EV71 virus by a dose dependent (MOI = 5, 10, 20) for 24 h. Using the LPS (1 μg/ml) for 6 h and 2 μM Nigericin for 30 min as a positive control. Supernatants were analyzed by ELISA for IL-1β secretion (D). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific for pro-IL-1β, pro-Caspase-1, and EV71 3D protein and GAPDH (Lys) (E). The mRNA levels for the gene pro-IL-1β were quantified by real-time PCR (F). Data shown are means ± SEM, *p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) The concentrations of IL-1β in culture supernatants were measured by ELISA kit (BD Biosciences, San Jose, CA).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Positive Control

    EV71 activates IL-1β through regulating the components of NLRP3 inflammasome complex. ( A and B ) HEK293T cells were transfected with plasmids encoding pro- IL-1β, Flag-pro-caspsase-1, Flag-NLRP3, and Flag-ASC. EV71 (MOI = 1 or 2) infected the transfected cells for 24 h. 5 mM ATP for 30 min as a positive control. Supernatants were analyzed by ELISA for IL-1β secretion (A). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to pro-IL-1β, pro-caspase-1, EV71 3D protein and GAPDH (Lys) (B). ( C and D ) TPA-differentiated THP-1 cells with shRNA-mediated specific gene silencing were stimulated by 2 μM Nigericin for 2 h. Supernatants were analyzed by ELISA for IL-1β secretion (C). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to IL-1β, Caspase-1, NLRP3, ASC, and GAPDH (Lys) (D). ( E and F ) TPA-differentiated THP-1 cells with shRNA-mediated knockdown of gene expression were infected by EV71 (MOI = 20) for 24 h. Supernatants were analyzed by ELISA for IL-1β secretion (E). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to IL-1β, Caspase-1, NLRP3, ASC, EV71 3D protein, and GAPDH (Lys) (F). Data shown are means±SEM, **p

    Journal: PLoS Pathogens

    Article Title: EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex

    doi: 10.1371/journal.ppat.1006123

    Figure Lengend Snippet: EV71 activates IL-1β through regulating the components of NLRP3 inflammasome complex. ( A and B ) HEK293T cells were transfected with plasmids encoding pro- IL-1β, Flag-pro-caspsase-1, Flag-NLRP3, and Flag-ASC. EV71 (MOI = 1 or 2) infected the transfected cells for 24 h. 5 mM ATP for 30 min as a positive control. Supernatants were analyzed by ELISA for IL-1β secretion (A). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to pro-IL-1β, pro-caspase-1, EV71 3D protein and GAPDH (Lys) (B). ( C and D ) TPA-differentiated THP-1 cells with shRNA-mediated specific gene silencing were stimulated by 2 μM Nigericin for 2 h. Supernatants were analyzed by ELISA for IL-1β secretion (C). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to IL-1β, Caspase-1, NLRP3, ASC, and GAPDH (Lys) (D). ( E and F ) TPA-differentiated THP-1 cells with shRNA-mediated knockdown of gene expression were infected by EV71 (MOI = 20) for 24 h. Supernatants were analyzed by ELISA for IL-1β secretion (E). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to IL-1β, Caspase-1, NLRP3, ASC, EV71 3D protein, and GAPDH (Lys) (F). Data shown are means±SEM, **p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) The concentrations of IL-1β in culture supernatants were measured by ELISA kit (BD Biosciences, San Jose, CA).

    Techniques: Transfection, Infection, Positive Control, Enzyme-linked Immunosorbent Assay, shRNA, Expressing

    The replication of EV71 virus is required for the activation of IL-1β mediated by NLRP3. ( A and B ) TPA-differentiated THP-1 cells were incubated by inactivated (UV or HOT) or live EV71 virus (MOI = 20) for 48 h. The LPS (1 μg/ml) for 6 h and 2 μM Nigericin for 30 min were used as positive controls. Supernatants were used for detection of IL-1β secretion by ELISA (A). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to pro-IL-1β, pro-caspase-1, EV71 3D protein and GAPDH (Lys) (B). ( C and D ) Human PBMCs were treated with LPS (1 μg/ml) was added for 6 h, inoculated with UV- or heat-inactivated EV71 (MOI = 5) for 36 h, or infected with infectious EV71 (MOI = 5, 10) for 36 h. Supernatants were used for detection IL-1β secretion by ELISA (C). The mRNA levels of pro-IL-1β were quantified by real-time PCR (D). ( E and F ) TPA-differentiated THP-1 cells were stimulated for 6 h with Lipo (Control), EV71 viral RNA (5 μg/ml) plus Lipo, HCV viral RNA (5 μg/ml) plus Lipo or 5 μg/ml poly(dA:dT) plus Lipo (positive control). Supernatants were used for detection IL-1β secretion by ELISA (E). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to pro-IL-1β, pro-caspase-1, and GAPDH (Lys) (F). Data shown are means±SEM, *p

    Journal: PLoS Pathogens

    Article Title: EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex

    doi: 10.1371/journal.ppat.1006123

    Figure Lengend Snippet: The replication of EV71 virus is required for the activation of IL-1β mediated by NLRP3. ( A and B ) TPA-differentiated THP-1 cells were incubated by inactivated (UV or HOT) or live EV71 virus (MOI = 20) for 48 h. The LPS (1 μg/ml) for 6 h and 2 μM Nigericin for 30 min were used as positive controls. Supernatants were used for detection of IL-1β secretion by ELISA (A). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to pro-IL-1β, pro-caspase-1, EV71 3D protein and GAPDH (Lys) (B). ( C and D ) Human PBMCs were treated with LPS (1 μg/ml) was added for 6 h, inoculated with UV- or heat-inactivated EV71 (MOI = 5) for 36 h, or infected with infectious EV71 (MOI = 5, 10) for 36 h. Supernatants were used for detection IL-1β secretion by ELISA (C). The mRNA levels of pro-IL-1β were quantified by real-time PCR (D). ( E and F ) TPA-differentiated THP-1 cells were stimulated for 6 h with Lipo (Control), EV71 viral RNA (5 μg/ml) plus Lipo, HCV viral RNA (5 μg/ml) plus Lipo or 5 μg/ml poly(dA:dT) plus Lipo (positive control). Supernatants were used for detection IL-1β secretion by ELISA (E). Immunoblot analysis of the mature (p17) form of IL-1β and cleaved caspase-1 in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to pro-IL-1β, pro-caspase-1, and GAPDH (Lys) (F). Data shown are means±SEM, *p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) The concentrations of IL-1β in culture supernatants were measured by ELISA kit (BD Biosciences, San Jose, CA).

    Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Infection, Real-time Polymerase Chain Reaction, Positive Control

    HTLV-1-induced LTB4 secretion depends on Tax-induced cPLA2γ overexpression. ( a ) Tax-expressing Jurkat cells secrete significant levels of LTB4. Jurkat cells were transduced with lentiviral vectors encoding GFP alone or together with the viral regulatory proteins Tax and HBZ. Protein expression was detected by western blot 48 h post transduction (right panel). Spontaneous LTB4 secretion levels were determined by ELISA. P value

    Journal: Nature Communications

    Article Title: HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation

    doi: 10.1038/ncomms15890

    Figure Lengend Snippet: HTLV-1-induced LTB4 secretion depends on Tax-induced cPLA2γ overexpression. ( a ) Tax-expressing Jurkat cells secrete significant levels of LTB4. Jurkat cells were transduced with lentiviral vectors encoding GFP alone or together with the viral regulatory proteins Tax and HBZ. Protein expression was detected by western blot 48 h post transduction (right panel). Spontaneous LTB4 secretion levels were determined by ELISA. P value

    Article Snippet: LTB4 was then measured using an ELISA kit (Cayman Chemical).

    Techniques: Over Expression, Expressing, Transduction, Western Blot, Enzyme-linked Immunosorbent Assay

    LTB4 secretion by HTLV-1-infected cells promote lymphocyte chemotaxis. ( a ) Drugs targeting the LTB4 synthesis pathway are efficient on C91/PL cells. C91/PL cells were pretreated with 200 nM MK886 or 1 μM MAFP (or left untreated, UT) for 30 min. Spontaneous secretion was determined by ELISA. P value

    Journal: Nature Communications

    Article Title: HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation

    doi: 10.1038/ncomms15890

    Figure Lengend Snippet: LTB4 secretion by HTLV-1-infected cells promote lymphocyte chemotaxis. ( a ) Drugs targeting the LTB4 synthesis pathway are efficient on C91/PL cells. C91/PL cells were pretreated with 200 nM MK886 or 1 μM MAFP (or left untreated, UT) for 30 min. Spontaneous secretion was determined by ELISA. P value

    Article Snippet: LTB4 was then measured using an ELISA kit (Cayman Chemical).

    Techniques: Infection, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay

    Blocking LTB4 synthesis during neoinfection in vivo results in lower HTLV-1 PVLs and a decrease in the number of infected clones. ( a ) Experimental procedure. Mice were considered humanized 8–10 weeks after CD34 + cell transplantation. Mice were then treated with IP injection of MK886 (5 nmol per mouse) three times a week. Injection with DMSO was used as control. One week after the initial injection, lethally irradiated MT2 cells were injected intraperitoneally. Six weeks after infection, mice were killed. Together, 14 mice were used. ( b ) LTB4 is increased in the plasma of HTLV-1-infected humanized mice. Six weeks after infection, plasma was collected and LTB4 levels were determined by ELISA. P value=0.006; bar represents mean. Kruskal–Wallis test. ( c ) The frequency of human splenocytes expressing CD25 is lower in infected animals treated with MK886. Six weeks after infection, mice were killed and splenocytes were collected. The percentage of human CD4 T cells (that is, hCD45 + ) positive for CD25 was determined by flow cytometry. P value=0.018; bar represents mean. Kruskal–Wallis test. ( d ) The PVL is lower in HTLV-1-infected mice treated with 5 nmol MK886. PVL is reported as the number of genomic tax copies per 100 human cells on splenocytes. P value=0.029; bar represents mean. Mann–Whitney U -test. ( e ) The number of UIS is lower in HTLV-1-infected mice treated with MK886. The number of independent HTLV-1-infected clones was determined by HTS clonality analysis ( Supplementary Table 1 ). P value=0.028; bar represents mean. Mann–Whitney U -test. ( f ) The abundance of infected clones is not altered upon MK886 treatment. Abundant and non-abundant clones, defined by ≥2 or a single sequencing read, respectively, were determined for each animal by iterative subsampling ( N =1,000) of an equivalent number of reads ( n =59, Supplementary Table 1 ) within the total read number that supported LTR-host junctions. Dot plots represent the distribution of the percentage of abundant clones (number of abundant clones/total number of UIS) over 1,000 subsampling iterations for each animal. P value=0.48; Median, quartile and 10–90th percentile are presented in the Box-and-Whisker plot. Mann–Whitney U -test.

    Journal: Nature Communications

    Article Title: HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation

    doi: 10.1038/ncomms15890

    Figure Lengend Snippet: Blocking LTB4 synthesis during neoinfection in vivo results in lower HTLV-1 PVLs and a decrease in the number of infected clones. ( a ) Experimental procedure. Mice were considered humanized 8–10 weeks after CD34 + cell transplantation. Mice were then treated with IP injection of MK886 (5 nmol per mouse) three times a week. Injection with DMSO was used as control. One week after the initial injection, lethally irradiated MT2 cells were injected intraperitoneally. Six weeks after infection, mice were killed. Together, 14 mice were used. ( b ) LTB4 is increased in the plasma of HTLV-1-infected humanized mice. Six weeks after infection, plasma was collected and LTB4 levels were determined by ELISA. P value=0.006; bar represents mean. Kruskal–Wallis test. ( c ) The frequency of human splenocytes expressing CD25 is lower in infected animals treated with MK886. Six weeks after infection, mice were killed and splenocytes were collected. The percentage of human CD4 T cells (that is, hCD45 + ) positive for CD25 was determined by flow cytometry. P value=0.018; bar represents mean. Kruskal–Wallis test. ( d ) The PVL is lower in HTLV-1-infected mice treated with 5 nmol MK886. PVL is reported as the number of genomic tax copies per 100 human cells on splenocytes. P value=0.029; bar represents mean. Mann–Whitney U -test. ( e ) The number of UIS is lower in HTLV-1-infected mice treated with MK886. The number of independent HTLV-1-infected clones was determined by HTS clonality analysis ( Supplementary Table 1 ). P value=0.028; bar represents mean. Mann–Whitney U -test. ( f ) The abundance of infected clones is not altered upon MK886 treatment. Abundant and non-abundant clones, defined by ≥2 or a single sequencing read, respectively, were determined for each animal by iterative subsampling ( N =1,000) of an equivalent number of reads ( n =59, Supplementary Table 1 ) within the total read number that supported LTR-host junctions. Dot plots represent the distribution of the percentage of abundant clones (number of abundant clones/total number of UIS) over 1,000 subsampling iterations for each animal. P value=0.48; Median, quartile and 10–90th percentile are presented in the Box-and-Whisker plot. Mann–Whitney U -test.

    Article Snippet: LTB4 was then measured using an ELISA kit (Cayman Chemical).

    Techniques: Blocking Assay, In Vivo, Infection, Clone Assay, Mouse Assay, Transplantation Assay, Injection, Irradiation, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry, MANN-WHITNEY, Sequencing, Whisker Assay

    Primary CD4 T cells from HTLV-1-infected individuals secrete LTB4. ( a ) PBMCs from HTLV-1-infected individuals secrete significantly more LTB4 than PBMCs from HDs. PBMCs were culture for 30 min and spontaneous LTB4 secretion levels were determined by ELISA. Dashed line represents the detection limit of the ELISA kit. PBMCs were from HDs ( n =5), HACs ( n =5) or individuals who had developed HAM/TSP ( n =5). Bars represent mean. P value=0.03; Mann–Whitney U -test (HDs versus HACs and HAM/TSP). ( b ) CD4 T cells from HTLV-1-infected individuals secrete detectable levels of LTB4. CD4 T cells were isolated, cultured for 30 min and spontaneous LTB4 secretion was detected by ELISA. Bars represent mean. P value

    Journal: Nature Communications

    Article Title: HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation

    doi: 10.1038/ncomms15890

    Figure Lengend Snippet: Primary CD4 T cells from HTLV-1-infected individuals secrete LTB4. ( a ) PBMCs from HTLV-1-infected individuals secrete significantly more LTB4 than PBMCs from HDs. PBMCs were culture for 30 min and spontaneous LTB4 secretion levels were determined by ELISA. Dashed line represents the detection limit of the ELISA kit. PBMCs were from HDs ( n =5), HACs ( n =5) or individuals who had developed HAM/TSP ( n =5). Bars represent mean. P value=0.03; Mann–Whitney U -test (HDs versus HACs and HAM/TSP). ( b ) CD4 T cells from HTLV-1-infected individuals secrete detectable levels of LTB4. CD4 T cells were isolated, cultured for 30 min and spontaneous LTB4 secretion was detected by ELISA. Bars represent mean. P value

    Article Snippet: LTB4 was then measured using an ELISA kit (Cayman Chemical).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Isolation, Cell Culture

    Koumine (KM) enhances LPS-induced astrocyte autophagy and attenuates astrocyte activation, as well as inflammation response in LPS-exposed primary astrocytes. Cultured astrocytes were exposed to koumine (0, 25, 50, or 100 μM) for 12 h prior to exposure to LPS for an additional 24 h before testing. Astrocytes in the control group were incubated with standard culture medium lacking koumine and LPS. Western blot (A) and densitometric quantification of GFAP (B) , Beclin 1 (C) , LC3-I/LC3-II (D) , P62 (E) , and GAPDH were performed in LPS-exposed astrocytes. Elisa were performed to explore the LPS-induced production of TNF-α (F) and IL-1β (G) in cultured astrocytes. Data are presented as the mean ± SEM. of three independent experiments. ### P

    Journal: Frontiers in Pharmacology

    Article Title: Koumine Decreases Astrocyte-Mediated Neuroinflammation and Enhances Autophagy, Contributing to Neuropathic Pain From Chronic Constriction Injury in Rats

    doi: 10.3389/fphar.2018.00989

    Figure Lengend Snippet: Koumine (KM) enhances LPS-induced astrocyte autophagy and attenuates astrocyte activation, as well as inflammation response in LPS-exposed primary astrocytes. Cultured astrocytes were exposed to koumine (0, 25, 50, or 100 μM) for 12 h prior to exposure to LPS for an additional 24 h before testing. Astrocytes in the control group were incubated with standard culture medium lacking koumine and LPS. Western blot (A) and densitometric quantification of GFAP (B) , Beclin 1 (C) , LC3-I/LC3-II (D) , P62 (E) , and GAPDH were performed in LPS-exposed astrocytes. Elisa were performed to explore the LPS-induced production of TNF-α (F) and IL-1β (G) in cultured astrocytes. Data are presented as the mean ± SEM. of three independent experiments. ### P

    Article Snippet: Total TNF-α, IL-6, and IL-1β levels were measured using ELISA Kits (Abcam, ab46070 for TNF-α and ab100768 for IL-1β, Cambridge, MA, United States) according to the manufacturer’s instructions.

    Techniques: Activation Assay, Cell Culture, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

    Letrozole reduced 17β-estradiol levels in the brain and shortened seizure latency. Mice were fed a diet containing cottonseed oil or cottonseed oil supplemented with DHA and injected with letrozole (10 mg/kg) intraperitoneally every other day for 28 days. ( a ) The 17β-estradiol levels in the cerebral cortex were determined by EIA. The values represent the mean ± S.E. (n = 7 animals in each group). Data were analyzed using Student’s t -test. ** P

    Journal: Scientific Reports

    Article Title: Potentiation of 17β-estradiol synthesis in the brain and elongation of seizure latency through dietary supplementation with docosahexaenoic acid

    doi: 10.1038/s41598-017-06630-0

    Figure Lengend Snippet: Letrozole reduced 17β-estradiol levels in the brain and shortened seizure latency. Mice were fed a diet containing cottonseed oil or cottonseed oil supplemented with DHA and injected with letrozole (10 mg/kg) intraperitoneally every other day for 28 days. ( a ) The 17β-estradiol levels in the cerebral cortex were determined by EIA. The values represent the mean ± S.E. (n = 7 animals in each group). Data were analyzed using Student’s t -test. ** P

    Article Snippet: The purified 17β-estradiol was quantified using EIA kit (Cayman Chemical, Ann Arbor, MI, USA).

    Techniques: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    Increased 17β-estradiol levels in the brain by DHA dietary supplementation. Mice were fed a diet containing soybean oil, cottonseed oil or cottonseed oil supplemented with DHA for 28 days. The cerebral cortex was then removed, and the 17β-estradiol levels were determined by EIA. The values represent the mean ± S.E. (n = 7 animals in each group). Data were analyzed using a one-way ANOVA, followed by Tukey’s test. * P

    Journal: Scientific Reports

    Article Title: Potentiation of 17β-estradiol synthesis in the brain and elongation of seizure latency through dietary supplementation with docosahexaenoic acid

    doi: 10.1038/s41598-017-06630-0

    Figure Lengend Snippet: Increased 17β-estradiol levels in the brain by DHA dietary supplementation. Mice were fed a diet containing soybean oil, cottonseed oil or cottonseed oil supplemented with DHA for 28 days. The cerebral cortex was then removed, and the 17β-estradiol levels were determined by EIA. The values represent the mean ± S.E. (n = 7 animals in each group). Data were analyzed using a one-way ANOVA, followed by Tukey’s test. * P

    Article Snippet: The purified 17β-estradiol was quantified using EIA kit (Cayman Chemical, Ann Arbor, MI, USA).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Ntn4 was overexpressed in GCs and negatively correlated with patient survival A. IHC staining of human GC tissues using Ntn4-specific antibody, as described in Methods; Magnification, × 200. B–C. Classification of samples according to the intensity of staining of Ntn4 expression ( n = 82, p = 0.001). D. ELISA analysis to determine serum level of Ntn4 in GC patients and normal controls ( n = 52, p

    Journal: Oncotarget

    Article Title: Netrin-4 as a biomarker promotes cell proliferation and invasion in gastric cancer

    doi:

    Figure Lengend Snippet: Ntn4 was overexpressed in GCs and negatively correlated with patient survival A. IHC staining of human GC tissues using Ntn4-specific antibody, as described in Methods; Magnification, × 200. B–C. Classification of samples according to the intensity of staining of Ntn4 expression ( n = 82, p = 0.001). D. ELISA analysis to determine serum level of Ntn4 in GC patients and normal controls ( n = 52, p

    Article Snippet: Quantification of cell supernatant and serum Nnt4 by ELISA One hundred microliters of cell supernatant or serum was used for the Ntn4 assay, which used an ELISA kit (Enzo Life Science Inc, USA).

    Techniques: Immunohistochemistry, Staining, Expressing, Enzyme-linked Immunosorbent Assay

    Overexpression of Ntn4 promoted the growth, migration and invasion of GC cells A–B. The overexpression of Ntn4 was achieved in AGS and MGC cells by transient transfection of pcDNA3-Ntn4 for 48 hours and with pcDNA3 vector (pcDNA3) only as control. Expression of Ntn4 was measured with Q-PCR (A) and ELISA (B). C–D. The enhanced expression of Ntn4 accelerated the growth of AGS (C) and MGC803 (D) cells. The cell proliferation assay was performed with CCK8 assay at 24, 48 and 72 hours, respectively after the transfection with pcDNA3 or pcDNA3-Ntn4. E. The enhanced expression of Ntn4 boosted the wound healing of AGS and MGC803 cells. The monolayer of cells transfected with pcDNA3 or pcDNA3-Ntn4 was disrupted with a tip and photographed under microscope at 0 and 24 hours; Magnification, × 100. F–G. The enhanced expression of Ntn4 promoted the invasion of AGS (F) and MGC803 (G) cells. Cells were transfected with pcDNA3 or pcDNA3-Ntn4 for 48 hours and subjected to the invasion assay; Magnification, × 100. Mean ± SEM, n ≥ 3. ** p

    Journal: Oncotarget

    Article Title: Netrin-4 as a biomarker promotes cell proliferation and invasion in gastric cancer

    doi:

    Figure Lengend Snippet: Overexpression of Ntn4 promoted the growth, migration and invasion of GC cells A–B. The overexpression of Ntn4 was achieved in AGS and MGC cells by transient transfection of pcDNA3-Ntn4 for 48 hours and with pcDNA3 vector (pcDNA3) only as control. Expression of Ntn4 was measured with Q-PCR (A) and ELISA (B). C–D. The enhanced expression of Ntn4 accelerated the growth of AGS (C) and MGC803 (D) cells. The cell proliferation assay was performed with CCK8 assay at 24, 48 and 72 hours, respectively after the transfection with pcDNA3 or pcDNA3-Ntn4. E. The enhanced expression of Ntn4 boosted the wound healing of AGS and MGC803 cells. The monolayer of cells transfected with pcDNA3 or pcDNA3-Ntn4 was disrupted with a tip and photographed under microscope at 0 and 24 hours; Magnification, × 100. F–G. The enhanced expression of Ntn4 promoted the invasion of AGS (F) and MGC803 (G) cells. Cells were transfected with pcDNA3 or pcDNA3-Ntn4 for 48 hours and subjected to the invasion assay; Magnification, × 100. Mean ± SEM, n ≥ 3. ** p

    Article Snippet: Quantification of cell supernatant and serum Nnt4 by ELISA One hundred microliters of cell supernatant or serum was used for the Ntn4 assay, which used an ELISA kit (Enzo Life Science Inc, USA).

    Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Expressing, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Proliferation Assay, CCK-8 Assay, Microscopy, Invasion Assay

    The growth-suppressive effect of Ntn4 knockdown on GC cells A–B. Ntn4 was efficiently knockdown by two independent Ntn4 siRNAs in AGS and MGC803 cells. Ntn4 expression was examined by Q-PCR (A) and ELISA (B) after transfection for 48 hours. C–D. Ntn4 ablation suppressed the proliferation of gastric cancer cells by CCK8 assays. The siRNAs were transfected into AGS (C) and MGC803 (D) cells as indicated for 24–72 hours. E. Ntn4 silencing reduced colony formation in AGS and MGC803 cells. Data are presented as mean ± SEM from three independent experiments with each running in triplicate. Unpaired student's t -test was used for the comparison between the two groups ( n = 3, * p

    Journal: Oncotarget

    Article Title: Netrin-4 as a biomarker promotes cell proliferation and invasion in gastric cancer

    doi:

    Figure Lengend Snippet: The growth-suppressive effect of Ntn4 knockdown on GC cells A–B. Ntn4 was efficiently knockdown by two independent Ntn4 siRNAs in AGS and MGC803 cells. Ntn4 expression was examined by Q-PCR (A) and ELISA (B) after transfection for 48 hours. C–D. Ntn4 ablation suppressed the proliferation of gastric cancer cells by CCK8 assays. The siRNAs were transfected into AGS (C) and MGC803 (D) cells as indicated for 24–72 hours. E. Ntn4 silencing reduced colony formation in AGS and MGC803 cells. Data are presented as mean ± SEM from three independent experiments with each running in triplicate. Unpaired student's t -test was used for the comparison between the two groups ( n = 3, * p

    Article Snippet: Quantification of cell supernatant and serum Nnt4 by ELISA One hundred microliters of cell supernatant or serum was used for the Ntn4 assay, which used an ELISA kit (Enzo Life Science Inc, USA).

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

    CFI bioactivity in a cohort of subjects with staged AMD and non-AMD normals. Subjects with AMD were staged according to the AREDS classification [ 24 ]. CFI bioactivity was determined by measuring the conversion of C3b to iC3b using ELISA for iC3b. Plasma dilutions (0.01–25 μl) were exposed to 80 μg/ml of each CFH and C3b and incubated at 37°C for 2 hours. Levels of iC3b were measured using ELISA. Samples from the same individual were run in the same assay as singlets. To control for the multiple assays conducted, each assay included a control sample. Data were fitted to a 4-parameter logistic equation and EC 50 s estimated. These values were converted to bioactivity in U/ μ g of CFI (1 mU = 1 / EC 50 in pg/ml). The logarithm of the bioactivity for the experimental samples and control plasma samples were analyzed by a mixed-model ANCOVA, using the control sample as a covariate to correct for inter-run variability. Data are shown as the fitted means in the original scale ± SE estimated from the fitted values using the Taylor series expansion. N = 4 to 47 patients per group. Legends: AREDS, Age-Related Eye Disease Study stages I through III, GA, geographic atrophy, Wet-Inactive, wet active AMD.

    Journal: PLoS ONE

    Article Title: A monoclonal antibody targeting amyloid β (Aβ) restores complement factor I bioactivity: Potential implications in age-related macular degeneration and Alzheimer’s disease

    doi: 10.1371/journal.pone.0195751

    Figure Lengend Snippet: CFI bioactivity in a cohort of subjects with staged AMD and non-AMD normals. Subjects with AMD were staged according to the AREDS classification [ 24 ]. CFI bioactivity was determined by measuring the conversion of C3b to iC3b using ELISA for iC3b. Plasma dilutions (0.01–25 μl) were exposed to 80 μg/ml of each CFH and C3b and incubated at 37°C for 2 hours. Levels of iC3b were measured using ELISA. Samples from the same individual were run in the same assay as singlets. To control for the multiple assays conducted, each assay included a control sample. Data were fitted to a 4-parameter logistic equation and EC 50 s estimated. These values were converted to bioactivity in U/ μ g of CFI (1 mU = 1 / EC 50 in pg/ml). The logarithm of the bioactivity for the experimental samples and control plasma samples were analyzed by a mixed-model ANCOVA, using the control sample as a covariate to correct for inter-run variability. Data are shown as the fitted means in the original scale ± SE estimated from the fitted values using the Taylor series expansion. N = 4 to 47 patients per group. Legends: AREDS, Age-Related Eye Disease Study stages I through III, GA, geographic atrophy, Wet-Inactive, wet active AMD.

    Article Snippet: ELISA detection of CFI, free Aβ and total bound Aβ CFI protein concentrations in human plasma were measured using a commercial ELISA kit from USCN Life Science (Wuhan, China), according to manufacturer’s instructions.

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation

    CFI bioactivity in plasma samples of Alzheimer’s disease (AD). Plasma was collected from the phase I study of AD receiving anti-amyloid β antibody GSK933776 at 6 mg/Kg. CFI bioactivity was determined by measuring the conversion of C3b to iC3b using ELISA for iC3b. Plasma dilutions (0.01–25 nl) were exposed to CFI and CFH (80 μg/ml each) and incubated for 2 hours at 37°C. Samples from same individuals were run in singlets within the same assay. Each assay included a control sample. Data were fitted using a non-linear mixed model that used a reparametrized[ 25 ] 4-parameter logistic equation and EC 50 s were estimated. The values were converted to bioactivity of CFI-Units/μg of CFI (1mU = 1/EC 50 in pg). The logarithm of CFI bioactivity for study and control samples were analyzed by a non-linear mixed effects model using the control sample as a covariate to correct for inter-run variability. Data are presented as the geometric means ± SE estimated using the Taylor series expansion (n = 3–5 per group). Percentages shown in the graph were calculated f rom the integrated areas under the curve for each dosing period using the trapezoidal rule on the geometric means.

    Journal: PLoS ONE

    Article Title: A monoclonal antibody targeting amyloid β (Aβ) restores complement factor I bioactivity: Potential implications in age-related macular degeneration and Alzheimer’s disease

    doi: 10.1371/journal.pone.0195751

    Figure Lengend Snippet: CFI bioactivity in plasma samples of Alzheimer’s disease (AD). Plasma was collected from the phase I study of AD receiving anti-amyloid β antibody GSK933776 at 6 mg/Kg. CFI bioactivity was determined by measuring the conversion of C3b to iC3b using ELISA for iC3b. Plasma dilutions (0.01–25 nl) were exposed to CFI and CFH (80 μg/ml each) and incubated for 2 hours at 37°C. Samples from same individuals were run in singlets within the same assay. Each assay included a control sample. Data were fitted using a non-linear mixed model that used a reparametrized[ 25 ] 4-parameter logistic equation and EC 50 s were estimated. The values were converted to bioactivity of CFI-Units/μg of CFI (1mU = 1/EC 50 in pg). The logarithm of CFI bioactivity for study and control samples were analyzed by a non-linear mixed effects model using the control sample as a covariate to correct for inter-run variability. Data are presented as the geometric means ± SE estimated using the Taylor series expansion (n = 3–5 per group). Percentages shown in the graph were calculated f rom the integrated areas under the curve for each dosing period using the trapezoidal rule on the geometric means.

    Article Snippet: ELISA detection of CFI, free Aβ and total bound Aβ CFI protein concentrations in human plasma were measured using a commercial ELISA kit from USCN Life Science (Wuhan, China), according to manufacturer’s instructions.

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation

    Anti-amyloid β (Aβ) antibody, GSK933776, effectively blocks Aβ’s ability to inhibit CFI bioactivity. Increasing doses of the antibody were preincubated with Aβ (30 μM) for 10 min at room temperature. CFI (1 μg/ml was added and incubated for 20 min at 37°C. CFH and C3b (80 μg/ml) were added and the mixture was incubated for an additional 30 min. Aliquots of the reaction mixture were analyzed for iC3b using ELISA. The curve in magenta shows the global fit of the two curves. The right dark red and dark blue bars show the effect of CFI + IgG1 (non-specific antibody control) on production of iC3b. The left 2 bars in lighter colors show the effect of CFI and IgG1 in presence of Aβ, i.e., reduction in CFI bioactivity. GSK933776 reduced Aβ’s ability to inhibit CFI in a dose-dependent manner (EC 50 ~ 20 μg/ml). Data are presented as means ± SEM of triplicate determinations for two experiments conducted over 2 separate weeks. A non-linear mixed effects model was used to estimate the EC 50 of the dose response curves using a reparametrized 4 parameter logistic equation [ 25 ]. Numbers in parentheses indicate 95% confidence intervals for the estimated EC 50 .

    Journal: PLoS ONE

    Article Title: A monoclonal antibody targeting amyloid β (Aβ) restores complement factor I bioactivity: Potential implications in age-related macular degeneration and Alzheimer’s disease

    doi: 10.1371/journal.pone.0195751

    Figure Lengend Snippet: Anti-amyloid β (Aβ) antibody, GSK933776, effectively blocks Aβ’s ability to inhibit CFI bioactivity. Increasing doses of the antibody were preincubated with Aβ (30 μM) for 10 min at room temperature. CFI (1 μg/ml was added and incubated for 20 min at 37°C. CFH and C3b (80 μg/ml) were added and the mixture was incubated for an additional 30 min. Aliquots of the reaction mixture were analyzed for iC3b using ELISA. The curve in magenta shows the global fit of the two curves. The right dark red and dark blue bars show the effect of CFI + IgG1 (non-specific antibody control) on production of iC3b. The left 2 bars in lighter colors show the effect of CFI and IgG1 in presence of Aβ, i.e., reduction in CFI bioactivity. GSK933776 reduced Aβ’s ability to inhibit CFI in a dose-dependent manner (EC 50 ~ 20 μg/ml). Data are presented as means ± SEM of triplicate determinations for two experiments conducted over 2 separate weeks. A non-linear mixed effects model was used to estimate the EC 50 of the dose response curves using a reparametrized 4 parameter logistic equation [ 25 ]. Numbers in parentheses indicate 95% confidence intervals for the estimated EC 50 .

    Article Snippet: ELISA detection of CFI, free Aβ and total bound Aβ CFI protein concentrations in human plasma were measured using a commercial ELISA kit from USCN Life Science (Wuhan, China), according to manufacturer’s instructions.

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    Preincubation of CFI with amyloid β (1–42) (Aβ) reduces CFI bioactivity. CFI (10–30 000 ng/ml) was incubated with Aβ (200 μM) for 1 hour at room temperature. CFH (10 μg/ml) and C3b (80 μg/ml) were added to the reaction and incubated at 37°C for 30 min. Aliquots of the reaction mixture were analyzed for iC3b by ELISA. Pre-incubation with Aβ causes a statistically significant shift of the curve to the right and approximately, a 5-fold decrease in CFI bioactivity. Data are presented as means ± SEM in triplicate wells. A non-linear mixed effects model was used to estimate the ratio of EC 50 for the dose response curves using a reparametrized 4 parameter logistic equation [ 25 ]. Number in parentheses indicate 95% confidence intervals for the estimated EC 50 ratio or EC 50 in ng/ml.

    Journal: PLoS ONE

    Article Title: A monoclonal antibody targeting amyloid β (Aβ) restores complement factor I bioactivity: Potential implications in age-related macular degeneration and Alzheimer’s disease

    doi: 10.1371/journal.pone.0195751

    Figure Lengend Snippet: Preincubation of CFI with amyloid β (1–42) (Aβ) reduces CFI bioactivity. CFI (10–30 000 ng/ml) was incubated with Aβ (200 μM) for 1 hour at room temperature. CFH (10 μg/ml) and C3b (80 μg/ml) were added to the reaction and incubated at 37°C for 30 min. Aliquots of the reaction mixture were analyzed for iC3b by ELISA. Pre-incubation with Aβ causes a statistically significant shift of the curve to the right and approximately, a 5-fold decrease in CFI bioactivity. Data are presented as means ± SEM in triplicate wells. A non-linear mixed effects model was used to estimate the ratio of EC 50 for the dose response curves using a reparametrized 4 parameter logistic equation [ 25 ]. Number in parentheses indicate 95% confidence intervals for the estimated EC 50 ratio or EC 50 in ng/ml.

    Article Snippet: ELISA detection of CFI, free Aβ and total bound Aβ CFI protein concentrations in human plasma were measured using a commercial ELISA kit from USCN Life Science (Wuhan, China), according to manufacturer’s instructions.

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    Measurement of CFI bioactivity. CFI bioactivity can be measured in plasma samples using an adapted assay that uses diluted plasma as the source of CFI. CFI bioactivity is temperature sensitive as heating the samples to 60° C abolishes the ability of the sample to generate iC3b (green symbol). Under the configuration of the assay, one can estimate the concentration of plasma that achieve half the amount of iC3b (i.e., 2.3 nl). We define this volume as 1 mU of CFI bioactivity. CFI bioactivity can be measured by determining the concentration of CFI protein in the same sample with an ELISA and correct the activity values by the amount of protein present in the sample, hence CFI bioactivity per unit of protein.

    Journal: PLoS ONE

    Article Title: A monoclonal antibody targeting amyloid β (Aβ) restores complement factor I bioactivity: Potential implications in age-related macular degeneration and Alzheimer’s disease

    doi: 10.1371/journal.pone.0195751

    Figure Lengend Snippet: Measurement of CFI bioactivity. CFI bioactivity can be measured in plasma samples using an adapted assay that uses diluted plasma as the source of CFI. CFI bioactivity is temperature sensitive as heating the samples to 60° C abolishes the ability of the sample to generate iC3b (green symbol). Under the configuration of the assay, one can estimate the concentration of plasma that achieve half the amount of iC3b (i.e., 2.3 nl). We define this volume as 1 mU of CFI bioactivity. CFI bioactivity can be measured by determining the concentration of CFI protein in the same sample with an ELISA and correct the activity values by the amount of protein present in the sample, hence CFI bioactivity per unit of protein.

    Article Snippet: ELISA detection of CFI, free Aβ and total bound Aβ CFI protein concentrations in human plasma were measured using a commercial ELISA kit from USCN Life Science (Wuhan, China), according to manufacturer’s instructions.

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    EREG stimulates leptin secretion and lipolysis in mice with diet-induced obesity (DIO). (A) DIO WT male mice (n=7/ group) were injected with 100μL PBS (Veh) with and without EREG (1.5ng/g body weight or 20ng/per iAb depot) into both epididymal iAb fat pads every other day for 2 weeks. Total mRNA was isolated from one whole iAb fad pad. Expression of Lep was measured using NanoString assay. Normalized data represent mean±SD, n=5; Mann-Whitney U test. (B) Expression of LEP protein levels were measured in plasma in the same mice group by ELISA. Data (mean±SD, n=5/group). Independent Student’s t -test. (C) Non-esterified fatty acids (NEFA) and (D) TG concentrations in plasma are shown as mean ± SD, n=5. Independent Student’s t -test.

    Journal: The Journal of endocrinology

    Article Title: Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice

    doi: 10.1530/JOE-18-0289

    Figure Lengend Snippet: EREG stimulates leptin secretion and lipolysis in mice with diet-induced obesity (DIO). (A) DIO WT male mice (n=7/ group) were injected with 100μL PBS (Veh) with and without EREG (1.5ng/g body weight or 20ng/per iAb depot) into both epididymal iAb fat pads every other day for 2 weeks. Total mRNA was isolated from one whole iAb fad pad. Expression of Lep was measured using NanoString assay. Normalized data represent mean±SD, n=5; Mann-Whitney U test. (B) Expression of LEP protein levels were measured in plasma in the same mice group by ELISA. Data (mean±SD, n=5/group). Independent Student’s t -test. (C) Non-esterified fatty acids (NEFA) and (D) TG concentrations in plasma are shown as mean ± SD, n=5. Independent Student’s t -test.

    Article Snippet: Media was collected after 2h, lyophilized and reconstituted in 120μL deionized water for LEP detection using ELISA kit (Alpco, Salem, NH).

    Techniques: Mouse Assay, Injection, Isolation, Expressing, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

    EREG/EGFR/MAPK axes induced leptin (LEP) secretion from adipose tissues ex vivo. Five iAb epididymal fat explants were excised from WT mice and stimulated. After 2h of stimulation LEP levels were measured in the media by ELISA. This experiment was repeated in three different WT mice. (A) Concentration-dependent secretion of LEP in the media from iAb fat explants stimulated with different concentrations of EREG. Data (mean±SD, n=5) are shown as percent to control (Veh, 100 %). Kruskal Wallis test. (B, C) LEP concentrations in the iAb fat lysates (B) and media (C) released from iAb fat before (triangles) and after (circles) stimulation with EREG (black bar, 50 ng/ml). Dashed and solid lines show mean value (n=3 mice) before and after stimulation. (D) Expression levels of Egfr and Erbb4 in epididymal iAb fat of lean WT mice (mean±SD, n=5) were measured by RT-PCR and normalized by TBP. (E) LEP concentrations in the iAb fat stimulated with EREG (black bar, 50 ng/ml) in the presence and absence of inhibitors of EGFR (10 μM) and MAPK (10 μM). Data (mean±SD, n=5) are shown as percent to control (Veh, white bar, 100%, dashed line). Between subject ANOVA with post-hoc Fisher LSD group comparison (α=0.05). (F) LEP in the media released following stimulation of mouse explants with and without EREG (50 ng/mL) in the presence and absence of inhibitors of HSL (10 μM), PPARα (10 μM), and PI3K (100 nM). Data (mean±SD, n=5) are shown as percent of control (Veh, 100%, dashed line). Between subject ANOVA. ns : not significant.

    Journal: The Journal of endocrinology

    Article Title: Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice

    doi: 10.1530/JOE-18-0289

    Figure Lengend Snippet: EREG/EGFR/MAPK axes induced leptin (LEP) secretion from adipose tissues ex vivo. Five iAb epididymal fat explants were excised from WT mice and stimulated. After 2h of stimulation LEP levels were measured in the media by ELISA. This experiment was repeated in three different WT mice. (A) Concentration-dependent secretion of LEP in the media from iAb fat explants stimulated with different concentrations of EREG. Data (mean±SD, n=5) are shown as percent to control (Veh, 100 %). Kruskal Wallis test. (B, C) LEP concentrations in the iAb fat lysates (B) and media (C) released from iAb fat before (triangles) and after (circles) stimulation with EREG (black bar, 50 ng/ml). Dashed and solid lines show mean value (n=3 mice) before and after stimulation. (D) Expression levels of Egfr and Erbb4 in epididymal iAb fat of lean WT mice (mean±SD, n=5) were measured by RT-PCR and normalized by TBP. (E) LEP concentrations in the iAb fat stimulated with EREG (black bar, 50 ng/ml) in the presence and absence of inhibitors of EGFR (10 μM) and MAPK (10 μM). Data (mean±SD, n=5) are shown as percent to control (Veh, white bar, 100%, dashed line). Between subject ANOVA with post-hoc Fisher LSD group comparison (α=0.05). (F) LEP in the media released following stimulation of mouse explants with and without EREG (50 ng/mL) in the presence and absence of inhibitors of HSL (10 μM), PPARα (10 μM), and PI3K (100 nM). Data (mean±SD, n=5) are shown as percent of control (Veh, 100%, dashed line). Between subject ANOVA. ns : not significant.

    Article Snippet: Media was collected after 2h, lyophilized and reconstituted in 120μL deionized water for LEP detection using ELISA kit (Alpco, Salem, NH).

    Techniques: Ex Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of the β2-GPI DNA vaccine and FK506 treatment on β2-GPI-specific Treg cell response. Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the percentage of Foxp3-expressing CD4+ T cells was determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± standard deviation (SD) of six mice from three independent experiments. (B) The culture supernatants were collected, and IL-10 and TGF-β production was analyzed in triplicate by ELISA. The data are presented as the means ± SD of triplicate assays from six mice/group. ns p > 0.05, **p

    Journal: PLoS ONE

    Article Title: Tolerogenic β2-glycoprotein I DNA vaccine and FK506 as an adjuvant attenuates experimental obstetric antiphospholipid syndrome

    doi: 10.1371/journal.pone.0198821

    Figure Lengend Snippet: Effects of the β2-GPI DNA vaccine and FK506 treatment on β2-GPI-specific Treg cell response. Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the percentage of Foxp3-expressing CD4+ T cells was determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± standard deviation (SD) of six mice from three independent experiments. (B) The culture supernatants were collected, and IL-10 and TGF-β production was analyzed in triplicate by ELISA. The data are presented as the means ± SD of triplicate assays from six mice/group. ns p > 0.05, **p

    Article Snippet: Anti-β2-GPI IgG concentrations were measured with an ELISA kit (MyBioSource, San Diego, CA) according to the manufacturer's protocol.

    Techniques: Purification, Recombinant, Expressing, Flow Cytometry, Cytometry, Standard Deviation, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Effects of the β2-GPI DNA vaccine and FK506 treatment on IgG anti-β2-GPI antibody levels. Serum samples were obtained from each mouse group on day 56, and IgG anti-β2-GPI antibody levels were analyzed by ELISA. The data are presented as the means ± standard deviation of triplicate assays from six mice/group. ns p > 0.05, *p

    Journal: PLoS ONE

    Article Title: Tolerogenic β2-glycoprotein I DNA vaccine and FK506 as an adjuvant attenuates experimental obstetric antiphospholipid syndrome

    doi: 10.1371/journal.pone.0198821

    Figure Lengend Snippet: Effects of the β2-GPI DNA vaccine and FK506 treatment on IgG anti-β2-GPI antibody levels. Serum samples were obtained from each mouse group on day 56, and IgG anti-β2-GPI antibody levels were analyzed by ELISA. The data are presented as the means ± standard deviation of triplicate assays from six mice/group. ns p > 0.05, *p

    Article Snippet: Anti-β2-GPI IgG concentrations were measured with an ELISA kit (MyBioSource, San Diego, CA) according to the manufacturer's protocol.

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Mouse Assay

    Effects of the β2-GPI DNA vaccine and FK506 treatment on cytokine production. Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the culture supernatants were collected, and interferon (IFN)- γ, interleukin (IL)-4 and IL-17A production was analyzed in triplicate by ELISA. The data are presented as the means ± standard deviation (SD) of triplicate assays from six mice/group. (B) The percentages of IFN-γ-expressing CD4+ T cells, IL-4-expressing CD4+ T cells and IL-17A-expressing CD4+ T cells were determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± SD of six mice from three independent experiments. ns p > 0.05, *p

    Journal: PLoS ONE

    Article Title: Tolerogenic β2-glycoprotein I DNA vaccine and FK506 as an adjuvant attenuates experimental obstetric antiphospholipid syndrome

    doi: 10.1371/journal.pone.0198821

    Figure Lengend Snippet: Effects of the β2-GPI DNA vaccine and FK506 treatment on cytokine production. Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the culture supernatants were collected, and interferon (IFN)- γ, interleukin (IL)-4 and IL-17A production was analyzed in triplicate by ELISA. The data are presented as the means ± standard deviation (SD) of triplicate assays from six mice/group. (B) The percentages of IFN-γ-expressing CD4+ T cells, IL-4-expressing CD4+ T cells and IL-17A-expressing CD4+ T cells were determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± SD of six mice from three independent experiments. ns p > 0.05, *p

    Article Snippet: Anti-β2-GPI IgG concentrations were measured with an ELISA kit (MyBioSource, San Diego, CA) according to the manufacturer's protocol.

    Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Standard Deviation, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    Comparison of serum soluble TLR2 (sTLR2) levels in the discovery cohort, validation cohort, and pooled data measured by enzyme-linked immunosorbent assay and analysis of pooled data. The data are presented as mean ± SD. (A) sTLR2 measured in the serum samples from RRMS patients and HC in the discovery cohort ( n = 21 RRMS, 24 HC). Mann–Whitney test showed significantly higher sTLR2 values in the RRMS patients ( P

    Journal: Frontiers in Immunology

    Article Title: The Soluble Form of Toll-Like Receptor 2 Is Elevated in Serum of Multiple Sclerosis Patients: A Novel Potential Disease Biomarker

    doi: 10.3389/fimmu.2018.00457

    Figure Lengend Snippet: Comparison of serum soluble TLR2 (sTLR2) levels in the discovery cohort, validation cohort, and pooled data measured by enzyme-linked immunosorbent assay and analysis of pooled data. The data are presented as mean ± SD. (A) sTLR2 measured in the serum samples from RRMS patients and HC in the discovery cohort ( n = 21 RRMS, 24 HC). Mann–Whitney test showed significantly higher sTLR2 values in the RRMS patients ( P

    Article Snippet: For the validation cohort sTLR2 was measured using a different ELISA kit (SEA753Hu; Cloud-Clone Corp., USA).

    Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Level of soluble TLR2 (sTLR2), sTLR4, and hsC-reactive protein (CRP) measured by enzyme-linked immunosorbent assay in the sera of relapsing-remitting (RR) MS (RRMS) patients and compared with HC. The data are presented as mean ± SD. (A) sTLR2 measured in the serum samples from RRMS patients during relapse ( n = 21) and compared with HC ( n = 24). Mann–Whitney test showed significantly higher sTLR2 values during relapse ( P

    Journal: Frontiers in Immunology

    Article Title: The Soluble Form of Toll-Like Receptor 2 Is Elevated in Serum of Multiple Sclerosis Patients: A Novel Potential Disease Biomarker

    doi: 10.3389/fimmu.2018.00457

    Figure Lengend Snippet: Level of soluble TLR2 (sTLR2), sTLR4, and hsC-reactive protein (CRP) measured by enzyme-linked immunosorbent assay in the sera of relapsing-remitting (RR) MS (RRMS) patients and compared with HC. The data are presented as mean ± SD. (A) sTLR2 measured in the serum samples from RRMS patients during relapse ( n = 21) and compared with HC ( n = 24). Mann–Whitney test showed significantly higher sTLR2 values during relapse ( P

    Article Snippet: For the validation cohort sTLR2 was measured using a different ELISA kit (SEA753Hu; Cloud-Clone Corp., USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Mass Spectrometry, MANN-WHITNEY

    Level of soluble TLR2 (sTLR2), sTLR4, and TLR2 stimulants in the urine of relapsing-remitting (RR) MS (RRMS) patients compared with HC. sTLR2 and sTLR4 were measured by enzyme-linked immunosorbent assay (ELISA) and urinary TLR2 stimulants were measured in human embryonic kidney (HEK)-293-TLR2 transfection system. (A) TLR2 stimulants in the urine of RRMS patients were measured in HEK-293-TLR2 transfectant system during relapse ( n = 18) and compared with HC ( n = 11). Mann–Whitney test showed a significantly higher TLR2 stimulants in the urine of MS patients during relapse ( P = 0.0004) compared with HC. Significantly higher urinary TLR2 stimulants were also observed during remission ( n = 13) compared to HC ( P = 0.0020) (not shown). (B) Wilcoxon matched-pairs signed rank test between relapse and remission also showed no significant difference ( n = 13, P = 0.0803). The values are expressed as ng/ml of the bacterial lipopeptide equivalent and the data are presented as mean ± SD. The data in Figure 3 (A,B) are log transformed from original values. (C) sTLR2 measured in the urine samples from RRMS patients during relapse ( n = 19) and HC ( n = 13). The data are presented as mean ± SD. Mann–Whitney test showed no significant differences between HC vs relapse ( P = 0.6498). HC vs remission ( n = 20) also showed no significant difference ( P = 0.2383) (not shown). (D) Wilcoxon matched-pairs signed rank test showed no significance between relapse and remission ( P = 0.3054). (E) sTLR4 measured in the urine samples from RRMS patients during relapse ( n = 17) and HC ( n = 10). The data are presented as mean ± SD. Mann–Whitney test showed no significant differences between HC vs relapse ( P = 0.1406). HC vs remission ( n = 14) also showed no significant difference ( P = 0.9290) (not shown). (F) Wilcoxon matched-pairs signed rank test showed no significance between relapse and remission ( n = 14, P = 0.1875). *** P

    Journal: Frontiers in Immunology

    Article Title: The Soluble Form of Toll-Like Receptor 2 Is Elevated in Serum of Multiple Sclerosis Patients: A Novel Potential Disease Biomarker

    doi: 10.3389/fimmu.2018.00457

    Figure Lengend Snippet: Level of soluble TLR2 (sTLR2), sTLR4, and TLR2 stimulants in the urine of relapsing-remitting (RR) MS (RRMS) patients compared with HC. sTLR2 and sTLR4 were measured by enzyme-linked immunosorbent assay (ELISA) and urinary TLR2 stimulants were measured in human embryonic kidney (HEK)-293-TLR2 transfection system. (A) TLR2 stimulants in the urine of RRMS patients were measured in HEK-293-TLR2 transfectant system during relapse ( n = 18) and compared with HC ( n = 11). Mann–Whitney test showed a significantly higher TLR2 stimulants in the urine of MS patients during relapse ( P = 0.0004) compared with HC. Significantly higher urinary TLR2 stimulants were also observed during remission ( n = 13) compared to HC ( P = 0.0020) (not shown). (B) Wilcoxon matched-pairs signed rank test between relapse and remission also showed no significant difference ( n = 13, P = 0.0803). The values are expressed as ng/ml of the bacterial lipopeptide equivalent and the data are presented as mean ± SD. The data in Figure 3 (A,B) are log transformed from original values. (C) sTLR2 measured in the urine samples from RRMS patients during relapse ( n = 19) and HC ( n = 13). The data are presented as mean ± SD. Mann–Whitney test showed no significant differences between HC vs relapse ( P = 0.6498). HC vs remission ( n = 20) also showed no significant difference ( P = 0.2383) (not shown). (D) Wilcoxon matched-pairs signed rank test showed no significance between relapse and remission ( P = 0.3054). (E) sTLR4 measured in the urine samples from RRMS patients during relapse ( n = 17) and HC ( n = 10). The data are presented as mean ± SD. Mann–Whitney test showed no significant differences between HC vs relapse ( P = 0.1406). HC vs remission ( n = 14) also showed no significant difference ( P = 0.9290) (not shown). (F) Wilcoxon matched-pairs signed rank test showed no significance between relapse and remission ( n = 14, P = 0.1875). *** P

    Article Snippet: For the validation cohort sTLR2 was measured using a different ELISA kit (SEA753Hu; Cloud-Clone Corp., USA).

    Techniques: Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Transfection, MANN-WHITNEY, Transformation Assay

    The construction of human ALIM infected by Streptococcus . A. The relative LDH vitality of supernates in ALIM at 4 h, 24 h and 48 h. B. The relative expression of IL-6, IL-8 and TNF-α of supernates in ALIM. LDH, lactate

    Journal: Bioengineered

    Article Title: Effect and mechanism of calpains on pediatric lobar pneumonia

    doi: 10.1080/21655979.2016.1234544

    Figure Lengend Snippet: The construction of human ALIM infected by Streptococcus . A. The relative LDH vitality of supernates in ALIM at 4 h, 24 h and 48 h. B. The relative expression of IL-6, IL-8 and TNF-α of supernates in ALIM. LDH, lactate

    Article Snippet: Quantitative measurement of IL-6 (RAB0306), IL-8 (RAB0319), and TNF-α (RAB1089) in supernates of ALIM was performed based on the human ELISA kit instructions (Sigma, USA).

    Techniques: Infection, Expressing

    The effect of calpains on the lung cells MRC-5. A. The transfection efficiency of siRNA-calpains 1 and 2 to lung fibroblast MRC-5 cells. B. Expressions of IL-6, IL-8 and TNF-α in transferred lung cells with calpains silence. C. The expressions

    Journal: Bioengineered

    Article Title: Effect and mechanism of calpains on pediatric lobar pneumonia

    doi: 10.1080/21655979.2016.1234544

    Figure Lengend Snippet: The effect of calpains on the lung cells MRC-5. A. The transfection efficiency of siRNA-calpains 1 and 2 to lung fibroblast MRC-5 cells. B. Expressions of IL-6, IL-8 and TNF-α in transferred lung cells with calpains silence. C. The expressions

    Article Snippet: Quantitative measurement of IL-6 (RAB0306), IL-8 (RAB0319), and TNF-α (RAB1089) in supernates of ALIM was performed based on the human ELISA kit instructions (Sigma, USA).

    Techniques: Transfection

    Evaluation of specificity and reactivity of six representative sdAbs against SIV-NP. Notes: ( A ) The sdAb of one representative member of each family extracted from IPTG-induced E. coli TG1 colonies was checked for their specific binding to the immobilized SIV-NP using an indirect ELISA. PEDV-N was used as an irrelevant protein control. ( B ) The reactivity of six representative sdAbs against SIV-NP was detected by indirect ELISA. Abbreviations: sdAb, single-domain antibody; IPTG, isopropyl-β-D-thiogalactopyranoside; SIV, swine influenza virus; NP, nucleoprotein; PEDV, porcine epidemic diarrhea virus; N, nucleocapsid.

    Journal: International Journal of Nanomedicine

    Article Title: Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

    doi: 10.2147/IJN.S218458

    Figure Lengend Snippet: Evaluation of specificity and reactivity of six representative sdAbs against SIV-NP. Notes: ( A ) The sdAb of one representative member of each family extracted from IPTG-induced E. coli TG1 colonies was checked for their specific binding to the immobilized SIV-NP using an indirect ELISA. PEDV-N was used as an irrelevant protein control. ( B ) The reactivity of six representative sdAbs against SIV-NP was detected by indirect ELISA. Abbreviations: sdAb, single-domain antibody; IPTG, isopropyl-β-D-thiogalactopyranoside; SIV, swine influenza virus; NP, nucleoprotein; PEDV, porcine epidemic diarrhea virus; N, nucleocapsid.

    Article Snippet: Evaluation of the Testing Consistency Among sdAb-ELISA, Commercial ELISA Kit, and Western Blot To compare consistency, sdAb-ELISA, the commercial ELISA kit (IDEXX Influenza A Ab Test, IDEXX Laboratories, Inc., Westbrook, ME, USA) and Western blot were used to detect 120 clinical swine serum samples at dilution of 1:10.

    Techniques: Binding Assay, Indirect ELISA

    Schematic representation of isolating sdAbs from an immunized camel and development of sdAb-based blocking ELISA. Abbreviation: sdAb, single-domain antibody.

    Journal: International Journal of Nanomedicine

    Article Title: Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

    doi: 10.2147/IJN.S218458

    Figure Lengend Snippet: Schematic representation of isolating sdAbs from an immunized camel and development of sdAb-based blocking ELISA. Abbreviation: sdAb, single-domain antibody.

    Article Snippet: Evaluation of the Testing Consistency Among sdAb-ELISA, Commercial ELISA Kit, and Western Blot To compare consistency, sdAb-ELISA, the commercial ELISA kit (IDEXX Influenza A Ab Test, IDEXX Laboratories, Inc., Westbrook, ME, USA) and Western blot were used to detect 120 clinical swine serum samples at dilution of 1:10.

    Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay

    Consistency between sdAb-ELISA and commercial ELISA kit. Notes: The 120 swine serum samples were detected using (A) the commercial ELISA kit and (B) sdAb-ELISA, respectively. Abbreviation: sdAb, single-domain antibody.

    Journal: International Journal of Nanomedicine

    Article Title: Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

    doi: 10.2147/IJN.S218458

    Figure Lengend Snippet: Consistency between sdAb-ELISA and commercial ELISA kit. Notes: The 120 swine serum samples were detected using (A) the commercial ELISA kit and (B) sdAb-ELISA, respectively. Abbreviation: sdAb, single-domain antibody.

    Article Snippet: Evaluation of the Testing Consistency Among sdAb-ELISA, Commercial ELISA Kit, and Western Blot To compare consistency, sdAb-ELISA, the commercial ELISA kit (IDEXX Influenza A Ab Test, IDEXX Laboratories, Inc., Westbrook, ME, USA) and Western blot were used to detect 120 clinical swine serum samples at dilution of 1:10.

    Techniques: Enzyme-linked Immunosorbent Assay

    Evaluation of the discrepancy of testing results between sdAb-ELISA and the commercial ELISA kit by Western blot. Notes: ( A ) Western blot was performed to detect swine serum samples with discrepancy between sdAb-ELISA and the commercial ELISA kit. ( B ) The detection results of three indicated methods. Abbreviation: sdAb, single-domain antibody.

    Journal: International Journal of Nanomedicine

    Article Title: Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

    doi: 10.2147/IJN.S218458

    Figure Lengend Snippet: Evaluation of the discrepancy of testing results between sdAb-ELISA and the commercial ELISA kit by Western blot. Notes: ( A ) Western blot was performed to detect swine serum samples with discrepancy between sdAb-ELISA and the commercial ELISA kit. ( B ) The detection results of three indicated methods. Abbreviation: sdAb, single-domain antibody.

    Article Snippet: Evaluation of the Testing Consistency Among sdAb-ELISA, Commercial ELISA Kit, and Western Blot To compare consistency, sdAb-ELISA, the commercial ELISA kit (IDEXX Influenza A Ab Test, IDEXX Laboratories, Inc., Westbrook, ME, USA) and Western blot were used to detect 120 clinical swine serum samples at dilution of 1:10.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

    Determination of the optimal conditions of sdAb-ELISA. Notes: ( A ) A checkerboard titration was used to test the optimal concentration range of biotinylated sdAb5 for each dilution of SIV-NP. ( B ) After the optimal concentration of biotinylated sdAb5 for each concentration SIV-NP was determined using another checkboard titration, the optimal dilution of serum was determined using sdAb-ELISA. Abbreviations: sdAb-ELISA, single-domain antibody-based blocking ELISA; SIV, swine influenza virus; NP, nucleoprotein.

    Journal: International Journal of Nanomedicine

    Article Title: Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

    doi: 10.2147/IJN.S218458

    Figure Lengend Snippet: Determination of the optimal conditions of sdAb-ELISA. Notes: ( A ) A checkerboard titration was used to test the optimal concentration range of biotinylated sdAb5 for each dilution of SIV-NP. ( B ) After the optimal concentration of biotinylated sdAb5 for each concentration SIV-NP was determined using another checkboard titration, the optimal dilution of serum was determined using sdAb-ELISA. Abbreviations: sdAb-ELISA, single-domain antibody-based blocking ELISA; SIV, swine influenza virus; NP, nucleoprotein.

    Article Snippet: Evaluation of the Testing Consistency Among sdAb-ELISA, Commercial ELISA Kit, and Western Blot To compare consistency, sdAb-ELISA, the commercial ELISA kit (IDEXX Influenza A Ab Test, IDEXX Laboratories, Inc., Westbrook, ME, USA) and Western blot were used to detect 120 clinical swine serum samples at dilution of 1:10.

    Techniques: Enzyme-linked Immunosorbent Assay, Titration, Concentration Assay, Blocking Assay

    Specificity and sensitivity of sdAb-ELISA. Notes: ( A ) The sera against SIV, PCV2, PEDV, PRRSV, PRV, and CSFV were used to evaluate specificity of the sdAb-ELISA. ( B ) The detection limit of the commercial ELISA kit (left) and sdAb-ELISA (right) were determined for an anti-SIV reference swine positive serum. ( C ) Serum samples were diluted 1/80 and used to evaluate and compare the sensitivity of the commercial ELISA kit (top) and sdAb-ELISA (bottom). Abbreviations: SIV, swine influenza virus; PCV2, porcine circovirus type 2; PEDV, porcine epidemic diarrhea virus; PRRSV, porcine reproductive and respiratory syndrome virus; PRV, pseudorabies virus; CSFV, classical swine fever virus.

    Journal: International Journal of Nanomedicine

    Article Title: Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

    doi: 10.2147/IJN.S218458

    Figure Lengend Snippet: Specificity and sensitivity of sdAb-ELISA. Notes: ( A ) The sera against SIV, PCV2, PEDV, PRRSV, PRV, and CSFV were used to evaluate specificity of the sdAb-ELISA. ( B ) The detection limit of the commercial ELISA kit (left) and sdAb-ELISA (right) were determined for an anti-SIV reference swine positive serum. ( C ) Serum samples were diluted 1/80 and used to evaluate and compare the sensitivity of the commercial ELISA kit (top) and sdAb-ELISA (bottom). Abbreviations: SIV, swine influenza virus; PCV2, porcine circovirus type 2; PEDV, porcine epidemic diarrhea virus; PRRSV, porcine reproductive and respiratory syndrome virus; PRV, pseudorabies virus; CSFV, classical swine fever virus.

    Article Snippet: Evaluation of the Testing Consistency Among sdAb-ELISA, Commercial ELISA Kit, and Western Blot To compare consistency, sdAb-ELISA, the commercial ELISA kit (IDEXX Influenza A Ab Test, IDEXX Laboratories, Inc., Westbrook, ME, USA) and Western blot were used to detect 120 clinical swine serum samples at dilution of 1:10.

    Techniques: Enzyme-linked Immunosorbent Assay

    EREG stimulates leptin secretion and lipolysis in mice with diet-induced obesity (DIO). (A) DIO WT male mice (n=7/ group) were injected with 100μL PBS (Veh) with and without EREG (1.5ng/g body weight or 20ng/per iAb depot) into both epididymal iAb fat pads every other day for 2 weeks. Total mRNA was isolated from one whole iAb fad pad. Expression of Lep was measured using NanoString assay. Normalized data represent mean±SD, n=5; Mann-Whitney U test. (B) Expression of LEP protein levels were measured in plasma in the same mice group by ELISA. Data (mean±SD, n=5/group). Independent Student’s t -test. (C) Non-esterified fatty acids (NEFA) and (D) TG concentrations in plasma are shown as mean ± SD, n=5. Independent Student’s t -test.

    Journal: The Journal of endocrinology

    Article Title: Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice

    doi: 10.1530/JOE-18-0289

    Figure Lengend Snippet: EREG stimulates leptin secretion and lipolysis in mice with diet-induced obesity (DIO). (A) DIO WT male mice (n=7/ group) were injected with 100μL PBS (Veh) with and without EREG (1.5ng/g body weight or 20ng/per iAb depot) into both epididymal iAb fat pads every other day for 2 weeks. Total mRNA was isolated from one whole iAb fad pad. Expression of Lep was measured using NanoString assay. Normalized data represent mean±SD, n=5; Mann-Whitney U test. (B) Expression of LEP protein levels were measured in plasma in the same mice group by ELISA. Data (mean±SD, n=5/group). Independent Student’s t -test. (C) Non-esterified fatty acids (NEFA) and (D) TG concentrations in plasma are shown as mean ± SD, n=5. Independent Student’s t -test.

    Article Snippet: Levels of LEP in iAb fat lysates produced and secreted into the medium were determined using ELISA kit (Crystal Chem, Elk Grove Village, IL).

    Techniques: Mouse Assay, Injection, Isolation, Expressing, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

    EREG/EGFR/MAPK axes induced leptin (LEP) secretion from adipose tissues ex vivo. Five iAb epididymal fat explants were excised from WT mice and stimulated. After 2h of stimulation LEP levels were measured in the media by ELISA. This experiment was repeated in three different WT mice. (A) Concentration-dependent secretion of LEP in the media from iAb fat explants stimulated with different concentrations of EREG. Data (mean±SD, n=5) are shown as percent to control (Veh, 100 %). Kruskal Wallis test. (B, C) LEP concentrations in the iAb fat lysates (B) and media (C) released from iAb fat before (triangles) and after (circles) stimulation with EREG (black bar, 50 ng/ml). Dashed and solid lines show mean value (n=3 mice) before and after stimulation. (D) Expression levels of Egfr and Erbb4 in epididymal iAb fat of lean WT mice (mean±SD, n=5) were measured by RT-PCR and normalized by TBP. (E) LEP concentrations in the iAb fat stimulated with EREG (black bar, 50 ng/ml) in the presence and absence of inhibitors of EGFR (10 μM) and MAPK (10 μM). Data (mean±SD, n=5) are shown as percent to control (Veh, white bar, 100%, dashed line). Between subject ANOVA with post-hoc Fisher LSD group comparison (α=0.05). (F) LEP in the media released following stimulation of mouse explants with and without EREG (50 ng/mL) in the presence and absence of inhibitors of HSL (10 μM), PPARα (10 μM), and PI3K (100 nM). Data (mean±SD, n=5) are shown as percent of control (Veh, 100%, dashed line). Between subject ANOVA. ns : not significant.

    Journal: The Journal of endocrinology

    Article Title: Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice

    doi: 10.1530/JOE-18-0289

    Figure Lengend Snippet: EREG/EGFR/MAPK axes induced leptin (LEP) secretion from adipose tissues ex vivo. Five iAb epididymal fat explants were excised from WT mice and stimulated. After 2h of stimulation LEP levels were measured in the media by ELISA. This experiment was repeated in three different WT mice. (A) Concentration-dependent secretion of LEP in the media from iAb fat explants stimulated with different concentrations of EREG. Data (mean±SD, n=5) are shown as percent to control (Veh, 100 %). Kruskal Wallis test. (B, C) LEP concentrations in the iAb fat lysates (B) and media (C) released from iAb fat before (triangles) and after (circles) stimulation with EREG (black bar, 50 ng/ml). Dashed and solid lines show mean value (n=3 mice) before and after stimulation. (D) Expression levels of Egfr and Erbb4 in epididymal iAb fat of lean WT mice (mean±SD, n=5) were measured by RT-PCR and normalized by TBP. (E) LEP concentrations in the iAb fat stimulated with EREG (black bar, 50 ng/ml) in the presence and absence of inhibitors of EGFR (10 μM) and MAPK (10 μM). Data (mean±SD, n=5) are shown as percent to control (Veh, white bar, 100%, dashed line). Between subject ANOVA with post-hoc Fisher LSD group comparison (α=0.05). (F) LEP in the media released following stimulation of mouse explants with and without EREG (50 ng/mL) in the presence and absence of inhibitors of HSL (10 μM), PPARα (10 μM), and PI3K (100 nM). Data (mean±SD, n=5) are shown as percent of control (Veh, 100%, dashed line). Between subject ANOVA. ns : not significant.

    Article Snippet: Levels of LEP in iAb fat lysates produced and secreted into the medium were determined using ELISA kit (Crystal Chem, Elk Grove Village, IL).

    Techniques: Ex Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    Tuft cells require Dclk1 expression to mediate its paracrine function. ( a ) Western blot analysis was performed to identify the protein expression level of COX2 in the vector control and DCLK1-overexpressing YAMC cells. ( b ) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to COX2. Lower bands represent IgG heavy and short chains. ( c ) Co-immunoprecipitation: IEC protein extracts from Dclk1 f/f mice at homeostasis and 24 h post-TBI were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to Cox2. Lower bands represent IgG heavy and short chains. ( d ) PGE2 EIA assay: Cell culture spent media collected and utilized for analyzing PGE2 levels. ( e ) Bar graph represents the percent colony formation of YAMC cells of vector control, Dclk1-overexpressing, PGE2-treated, and co-culture (YAMC + YAMC Dclk1-overexpression) cells at baseline and 48 h post-radiation. ( f ) Proposed Mechanism: Tuft cells regulates ATM mediated DDR in response to injury to enhance intestinal epithelial survival and self-renewal for effective restitution and function. In response to radiation-induced injury, Dclk1 + tuft cells enhance COX2 signaling for the paracrine regulation of IECs/ISCs. Dclk1 expression is critical for ATM activation for the DNA DSB repair response. ATM activation significantly controls DNA repair, apoptosis, and the cell cycle. Cell survival and self-renewal depends upon cellular DNA integrity. Dclk1 regulation of the ATM pathway enhances cell survival and self-renewal for effective intestinal regeneration after TBI.

    Journal: Scientific Reports

    Article Title: Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury

    doi: 10.1038/srep37667

    Figure Lengend Snippet: Tuft cells require Dclk1 expression to mediate its paracrine function. ( a ) Western blot analysis was performed to identify the protein expression level of COX2 in the vector control and DCLK1-overexpressing YAMC cells. ( b ) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to COX2. Lower bands represent IgG heavy and short chains. ( c ) Co-immunoprecipitation: IEC protein extracts from Dclk1 f/f mice at homeostasis and 24 h post-TBI were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to Cox2. Lower bands represent IgG heavy and short chains. ( d ) PGE2 EIA assay: Cell culture spent media collected and utilized for analyzing PGE2 levels. ( e ) Bar graph represents the percent colony formation of YAMC cells of vector control, Dclk1-overexpressing, PGE2-treated, and co-culture (YAMC + YAMC Dclk1-overexpression) cells at baseline and 48 h post-radiation. ( f ) Proposed Mechanism: Tuft cells regulates ATM mediated DDR in response to injury to enhance intestinal epithelial survival and self-renewal for effective restitution and function. In response to radiation-induced injury, Dclk1 + tuft cells enhance COX2 signaling for the paracrine regulation of IECs/ISCs. Dclk1 expression is critical for ATM activation for the DNA DSB repair response. ATM activation significantly controls DNA repair, apoptosis, and the cell cycle. Cell survival and self-renewal depends upon cellular DNA integrity. Dclk1 regulation of the ATM pathway enhances cell survival and self-renewal for effective intestinal regeneration after TBI.

    Article Snippet: PGE2 Measurement The PGE2 levels in the serum and spent cell culture media were measured using a PGE2 EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA).

    Techniques: Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay, Over Expression, Activation Assay