gene cdna  (Sino Biological)


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    Structured Review

    Sino Biological gene cdna
    Gene Cdna, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene cdna/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gene cdna - by Bioz Stars, 2021-07
    86/100 stars

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    Related Articles

    Mutagenesis:

    Article Title: N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer’s disease
    Article Snippet: Quantification was achieved by comparison with NBD-S1P (Avanti Polar Lipids) standards. .. Mutagenesis of the SphK1 and COX2 proteins Single point mutations in SphK1 (R56A, R57A, R24A, R185A, D178A, and F192A) and COX2 (S565A, N181A, T564A, and S567A) were generated using the In-Fusion Cloning Kits (Clontech) and ORF cDNA expression plasmids of human SphK1 and COX2 gene (Sino Biological Inc, HG15679-NH and HG12036-NH), according to the manufacturer’s instructions. .. The SphK1 WT, SphK1 mutants, COX2 WT, and COX2 mutant plasmids were transformed in the One shot TOP 10 competent cell (Invitrogen).

    Generated:

    Article Title: N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer’s disease
    Article Snippet: Quantification was achieved by comparison with NBD-S1P (Avanti Polar Lipids) standards. .. Mutagenesis of the SphK1 and COX2 proteins Single point mutations in SphK1 (R56A, R57A, R24A, R185A, D178A, and F192A) and COX2 (S565A, N181A, T564A, and S567A) were generated using the In-Fusion Cloning Kits (Clontech) and ORF cDNA expression plasmids of human SphK1 and COX2 gene (Sino Biological Inc, HG15679-NH and HG12036-NH), according to the manufacturer’s instructions. .. The SphK1 WT, SphK1 mutants, COX2 WT, and COX2 mutant plasmids were transformed in the One shot TOP 10 competent cell (Invitrogen).

    Article Title: Pathogenicity of two COQ7 mutations and responses to 2,4‐dihydroxybenzoate bypass treatment
    Article Snippet: .. Retroviral vectors and gene transferThe coding region for COQ7 was generated by PCR amplification from a full‐length human COQ7 cDNA (HG12195‐G; Sino Biological Inc., Beijing, China) and cloned into the BamH1 and EcoR1 sites of pBabe‐hygro (obtained from Addgene). .. The resulting retroviral expressing vector is named pBabe‐hCOQ7‐hygro.

    Clone Assay:

    Article Title: N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer’s disease
    Article Snippet: Quantification was achieved by comparison with NBD-S1P (Avanti Polar Lipids) standards. .. Mutagenesis of the SphK1 and COX2 proteins Single point mutations in SphK1 (R56A, R57A, R24A, R185A, D178A, and F192A) and COX2 (S565A, N181A, T564A, and S567A) were generated using the In-Fusion Cloning Kits (Clontech) and ORF cDNA expression plasmids of human SphK1 and COX2 gene (Sino Biological Inc, HG15679-NH and HG12036-NH), according to the manufacturer’s instructions. .. The SphK1 WT, SphK1 mutants, COX2 WT, and COX2 mutant plasmids were transformed in the One shot TOP 10 competent cell (Invitrogen).

    Article Title: Pathogenicity of two COQ7 mutations and responses to 2,4‐dihydroxybenzoate bypass treatment
    Article Snippet: .. Retroviral vectors and gene transferThe coding region for COQ7 was generated by PCR amplification from a full‐length human COQ7 cDNA (HG12195‐G; Sino Biological Inc., Beijing, China) and cloned into the BamH1 and EcoR1 sites of pBabe‐hygro (obtained from Addgene). .. The resulting retroviral expressing vector is named pBabe‐hCOQ7‐hygro.

    Article Title: CYR61 (CCN1) overexpression induces lung injury in mice
    Article Snippet: ELISA was developed in house to measure CYR61. .. To generate murine CYR61 standard, the cDNA of mouse Cyr61 (accession number NM 010516.2), was obtained from Sino Biological (Beijing, China) and cloned into a mammalian expression vector. .. The construct contains the whole Cyr61- coding cDNA, including the native start codon, and a 10-histidine tag on the 3′ end of the gene.

    Expressing:

    Article Title: N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer’s disease
    Article Snippet: Quantification was achieved by comparison with NBD-S1P (Avanti Polar Lipids) standards. .. Mutagenesis of the SphK1 and COX2 proteins Single point mutations in SphK1 (R56A, R57A, R24A, R185A, D178A, and F192A) and COX2 (S565A, N181A, T564A, and S567A) were generated using the In-Fusion Cloning Kits (Clontech) and ORF cDNA expression plasmids of human SphK1 and COX2 gene (Sino Biological Inc, HG15679-NH and HG12036-NH), according to the manufacturer’s instructions. .. The SphK1 WT, SphK1 mutants, COX2 WT, and COX2 mutant plasmids were transformed in the One shot TOP 10 competent cell (Invitrogen).

    Article Title: CYR61 (CCN1) overexpression induces lung injury in mice
    Article Snippet: ELISA was developed in house to measure CYR61. .. To generate murine CYR61 standard, the cDNA of mouse Cyr61 (accession number NM 010516.2), was obtained from Sino Biological (Beijing, China) and cloned into a mammalian expression vector. .. The construct contains the whole Cyr61- coding cDNA, including the native start codon, and a 10-histidine tag on the 3′ end of the gene.

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus
    Article Snippet: Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (Tree Star). .. Pseudotyped lentivirus production and titer measurement Lentivector plasmids containing RFP or GFP reporter genes were co-transfected with either SARS-CoV-2 spike protein or SARS -CoV spike protein (Human SARS coronavirus (SARS-CoV) spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized) from SinoBiological) plasmids into HEK-293T cells using Lipofectamine TM 3000 (Invitrogen) according to the manufacturer’s protocol. .. Viral supernatants were collected 24–48 h post-transfection, filtered through a 0.45 μm syringe filter (Millipore) to remove cellular debris, and concentrated with Lenti-X (Invitrogen) according to the manufacturers' protocol.

    Mouse Assay:

    Article Title: Type I Interferons Control Proliferation and Function of the Intestinal Epithelium
    Article Snippet: Intriguingly, a significant enrichment for the genes altered in the latter mice was seen upon the concurrent ablation of CK1α and IFNAR1 ( ). .. The mild hyperplasia of the small intestine ( ) and colonic epithelium ( ) as well as the progressive loss of goblet cells ( and ) observed in Csnk1a1 Δgut ; Ifnar1 −/− mice were indeed somewhat reminiscent of the phenotype in Apc Δgut ; Ifnar1 +/+ animals. .. Furthermore, Csnk1a1 Δgut ; Ifnar1 −/− mice rapidly became moribund within a week after tamoxifen treatment was completed ( ).

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice
    Article Snippet: The lungs were harvested and prepared for flow cytometric analyses after exposure of mice to CS for 7 days. .. For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group). .. Lung tissues were collected for western blot and enzyme-linked immunoassay (ELISA) analyses at 7 days after CS instillation.

    Polymerase Chain Reaction:

    Article Title: Pathogenicity of two COQ7 mutations and responses to 2,4‐dihydroxybenzoate bypass treatment
    Article Snippet: .. Retroviral vectors and gene transferThe coding region for COQ7 was generated by PCR amplification from a full‐length human COQ7 cDNA (HG12195‐G; Sino Biological Inc., Beijing, China) and cloned into the BamH1 and EcoR1 sites of pBabe‐hygro (obtained from Addgene). .. The resulting retroviral expressing vector is named pBabe‐hCOQ7‐hygro.

    Amplification:

    Article Title: Pathogenicity of two COQ7 mutations and responses to 2,4‐dihydroxybenzoate bypass treatment
    Article Snippet: .. Retroviral vectors and gene transferThe coding region for COQ7 was generated by PCR amplification from a full‐length human COQ7 cDNA (HG12195‐G; Sino Biological Inc., Beijing, China) and cloned into the BamH1 and EcoR1 sites of pBabe‐hygro (obtained from Addgene). .. The resulting retroviral expressing vector is named pBabe‐hCOQ7‐hygro.

    Plasmid Preparation:

    Article Title: CYR61 (CCN1) overexpression induces lung injury in mice
    Article Snippet: ELISA was developed in house to measure CYR61. .. To generate murine CYR61 standard, the cDNA of mouse Cyr61 (accession number NM 010516.2), was obtained from Sino Biological (Beijing, China) and cloned into a mammalian expression vector. .. The construct contains the whole Cyr61- coding cDNA, including the native start codon, and a 10-histidine tag on the 3′ end of the gene.

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus
    Article Snippet: Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (Tree Star). .. Pseudotyped lentivirus production and titer measurement Lentivector plasmids containing RFP or GFP reporter genes were co-transfected with either SARS-CoV-2 spike protein or SARS -CoV spike protein (Human SARS coronavirus (SARS-CoV) spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized) from SinoBiological) plasmids into HEK-293T cells using Lipofectamine TM 3000 (Invitrogen) according to the manufacturer’s protocol. .. Viral supernatants were collected 24–48 h post-transfection, filtered through a 0.45 μm syringe filter (Millipore) to remove cellular debris, and concentrated with Lenti-X (Invitrogen) according to the manufacturers' protocol.

    Injection:

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice
    Article Snippet: The lungs were harvested and prepared for flow cytometric analyses after exposure of mice to CS for 7 days. .. For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group). .. Lung tissues were collected for western blot and enzyme-linked immunoassay (ELISA) analyses at 7 days after CS instillation.

    SPR Assay:

    Article Title: Immune Repertoire Diversity Correlated with Mortality in Avian Influenza A (H7N9) Virus Infected Patients
    Article Snippet: Protein purity was estimated as > 90% by SDS–polyacrylamide gel electrophoresis, and protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare). .. Surface Plasmon Resonance binding experimentsThe binding experiments were performed using a ProteOn XRP36 system (Bio-Rad, Hercules, CA) to determine the kinetics of scFvs N2_3050, o1b1 and l1b1 to H7N9 HA antigen (Sino Biological Inc.). .. H7N9 HA was immobilized on the ProteOn GLM biosensor chip using standard amine coupling chemistry (300 nM in 10 mM sodium acetate buffer, pH 5.0).

    Binding Assay:

    Article Title: Immune Repertoire Diversity Correlated with Mortality in Avian Influenza A (H7N9) Virus Infected Patients
    Article Snippet: Protein purity was estimated as > 90% by SDS–polyacrylamide gel electrophoresis, and protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare). .. Surface Plasmon Resonance binding experimentsThe binding experiments were performed using a ProteOn XRP36 system (Bio-Rad, Hercules, CA) to determine the kinetics of scFvs N2_3050, o1b1 and l1b1 to H7N9 HA antigen (Sino Biological Inc.). .. H7N9 HA was immobilized on the ProteOn GLM biosensor chip using standard amine coupling chemistry (300 nM in 10 mM sodium acetate buffer, pH 5.0).

    Enzyme-linked Immunosorbent Assay:

    Article Title: In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections
    Article Snippet: Detection of Mouse Anti-Influenza mAbs C179, S139/1, and 9H10 For co-expression studies, to discriminate the expression of S139/1 and 9H10, both recognizing H3 epitopes, S139/1 was detected using the HA1 subunit and 9H10 was detected with H10N8. .. ELISA plates coated with 200 ng of H1N1 (A/WSN/33) HA, 50 ng of H3N2 (A/Aichi/2/1968) HA1 or HA subunit, or 200 ng of H10N8 (A/Jiangxi-Donghu/346/2013) HA (Sino Biologicals, Beijing, China) were blocked and incubated with serially diluted mouse serum. ..

    Incubation:

    Article Title: In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections
    Article Snippet: Detection of Mouse Anti-Influenza mAbs C179, S139/1, and 9H10 For co-expression studies, to discriminate the expression of S139/1 and 9H10, both recognizing H3 epitopes, S139/1 was detected using the HA1 subunit and 9H10 was detected with H10N8. .. ELISA plates coated with 200 ng of H1N1 (A/WSN/33) HA, 50 ng of H3N2 (A/Aichi/2/1968) HA1 or HA subunit, or 200 ng of H10N8 (A/Jiangxi-Donghu/346/2013) HA (Sino Biologicals, Beijing, China) were blocked and incubated with serially diluted mouse serum. ..

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    Sino Biological 4 1bb fusion protein treatment100
    Blockade of <t>4-1BB</t> signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.
    4 1bb Fusion Protein Treatment100, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    94
    Sino Biological h1n1
    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 <t>(H1N1)</t> or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p
    H1n1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h1n1/product/Sino Biological
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    99
    Sino Biological gfp reporter genes
    Neutralizing titers for SARS-CoV-2 and SARS-CoV in COVID-19 subject plasma. a Neutralization assay with S-RBD-specific NAb, healthy control plasma, and a COVID-19 patient plasma. Threefold serial dilutions of NAb from 10 μg/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with spike protein pseudovirus and added to 293-ACE2 cells. <t>GFP</t> expression was analyzed by flow cytometry 3 days post infection. b SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as an outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups ( n = 113). c NT50 of COVID-19 patient and plasma donor groups subdivided into males and females ( n = 113). d Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups ( n = 104), 293-ACE2 cells were infected and <t>RFP</t> expression was determined at day 3 using flow cytometry. e Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV ( n = 46). Two-tailed Mann–Whitney U test was used to determine the statistical significances in figures ( b ), ( c ) and ( d ) and two-tailed Spearman’s was used for figure ( e ). Horizontal bars in ( b ), ( c ) and ( d ) indicate mean values.
    Gfp Reporter Genes, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sino Biological sars cov 2 nucleoprotein
    <t>SARS-CoV-2</t> nucleoprotein was present in several tissues of COVID-19-negative donors. ( a ) Representative images of donor tissue specimens analyzed by Western blot. Labels in the images show the location of tissue sampling in each organ/tissue. ( b ) Western blot analysis for SARS-CoV-2 nucleoprotein on total protein extracts from tissues of 8 different deceased donor PVW specimens, RV specimens from healthy donors (collected prior to COVID-19 pandemic), and LV specimen from COVID-19-positive deceased patient. GAPDH was used as a loading control protein. ( c , d ) Western blot analysis for SARS-CoV-2 nucleoprotein on total protein extracts from different cardiac and valve tissues of 2 deceased donors (#20-22 and #20-24) positive for SARS-CoV-2 serological assay. RV specimen from a healthy donor (collected prior to COVID-19 pandemic) and LV specimen from a COVID-19-positive deceased patient were used as controls. GAPDH was used as the loading control protein. PVW: pulmonary vein wall; RA: right atrium; IAS: interatrial septum; LA: left atrium; RV: right ventricle; IVS: interventricular septum; LV: left ventricle; AW: aortic wall; AVL: aortic valve leaflet.
    Sars Cov 2 Nucleoprotein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    A model for alveolar macrophages (AMs) expressing 4-1BB in the regulation of pulmonary fibrosis through secreting pro-fibrotic mediators. The expression of 4-1BB increases in AMs in response to crystalline silica, which leads to elevated secretion of pro-inflammatory and pro-fibrotic cytokines, chemokines, and MMPs. These pro-fibrotic mediators promote pulmonary alveoli injury, the accumulation of monocytes, lymphocytes, and fibrocytes, and collagen deposition, resulting in pulmonary fibrosis.

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: A model for alveolar macrophages (AMs) expressing 4-1BB in the regulation of pulmonary fibrosis through secreting pro-fibrotic mediators. The expression of 4-1BB increases in AMs in response to crystalline silica, which leads to elevated secretion of pro-inflammatory and pro-fibrotic cytokines, chemokines, and MMPs. These pro-fibrotic mediators promote pulmonary alveoli injury, the accumulation of monocytes, lymphocytes, and fibrocytes, and collagen deposition, resulting in pulmonary fibrosis.

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Affinity Magnetic Separation, Expressing

    Expression of 4-1BB on CD4+ T cells. (A) Representative plots of flow cytometric analyses for 4-1BB on effector and naïve T cells. (B) The percentage of effector T cells expressing 4-1BB. (C,D) The frequency of effector and naïve T cells in CD4+ T cells from the lungs. (E) The percentage of naïve T cells expressing 4-1BB. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; ns, not significant).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Expression of 4-1BB on CD4+ T cells. (A) Representative plots of flow cytometric analyses for 4-1BB on effector and naïve T cells. (B) The percentage of effector T cells expressing 4-1BB. (C,D) The frequency of effector and naïve T cells in CD4+ T cells from the lungs. (E) The percentage of naïve T cells expressing 4-1BB. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; ns, not significant).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Expressing

    4-1BB expression on the mouse alveolar macrophage cell line, MH-S. MH-S cells were treated with crystalline silica (50 µg/cm 2 ) or saline for 12 h. (A,B) The percentage of MH-S cells expressing 4-1BB ( n = 4). (C) Western blot analysis of 4-1BB protein from whole cell lysates. (D) Quantification of the 4-1BB protein level relative to that of β-actin is shown ( n = 3). (E) Total RNA was isolated to analyze 4-1BB mRNA expression ( n = 4) relative to GAPDH. The data were representative of three independent experiments. Data were expressed as mean ± SEM (*** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: 4-1BB expression on the mouse alveolar macrophage cell line, MH-S. MH-S cells were treated with crystalline silica (50 µg/cm 2 ) or saline for 12 h. (A,B) The percentage of MH-S cells expressing 4-1BB ( n = 4). (C) Western blot analysis of 4-1BB protein from whole cell lysates. (D) Quantification of the 4-1BB protein level relative to that of β-actin is shown ( n = 3). (E) Total RNA was isolated to analyze 4-1BB mRNA expression ( n = 4) relative to GAPDH. The data were representative of three independent experiments. Data were expressed as mean ± SEM (*** p ≤ 0.001).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Expressing, Western Blot, Isolation

    Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

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

    The secretion of pro-fibrotic mediators is reduced in the lungs from crystalline silica (CS)-injured mice, upon inhibition of 4-1BB signaling. C57BL/6 mice were administered a CS suspension or saline, respectively; 4-1BBIg or isotype control (IgG1) were injected intraperitoneally (i.p.; n = 3–4). (A–D) Quantification of MMP9 and MMP12 protein levels by western blot, which were normalized to those of β-actin in lungs. Shown as bar graph. (E–H) ELISA analysis of cytokines in lung tissues. (E) IL-1β, (F) IL-6, (G) tumor necrosis factor-α, (H) monocyte chemoattractant protein-1. Experiments were performed three times. C57BL/6 mice were administered a CS suspension or saline, respectively; NQDI 1 or isotype control were injected i.p. ( n = 10). (I) Immunohistochemical staining of paraffin-embedded lung tissue sections at 7 and 56 days showed CD68, MMP9, and MMP12 expression. Nuclei were stained by hematoxylin (blue). (J–L) Identification of MMP9 and MMP12 protein levels in mouse lung tissues at 7 and 56 days by western blot. The levels of MMP9 and MMP12 were normalized to those of β-actin. (M) Representative images for the immunohistochemical staining of collagen I in paraffin-embedded lung tissue sections 56 days after CS instillation. Nuclei were stained by hematoxylin (blue). (I,M) Scale bar, 50 µm. Experiments were performed three times. Data are shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: The secretion of pro-fibrotic mediators is reduced in the lungs from crystalline silica (CS)-injured mice, upon inhibition of 4-1BB signaling. C57BL/6 mice were administered a CS suspension or saline, respectively; 4-1BBIg or isotype control (IgG1) were injected intraperitoneally (i.p.; n = 3–4). (A–D) Quantification of MMP9 and MMP12 protein levels by western blot, which were normalized to those of β-actin in lungs. Shown as bar graph. (E–H) ELISA analysis of cytokines in lung tissues. (E) IL-1β, (F) IL-6, (G) tumor necrosis factor-α, (H) monocyte chemoattractant protein-1. Experiments were performed three times. C57BL/6 mice were administered a CS suspension or saline, respectively; NQDI 1 or isotype control were injected i.p. ( n = 10). (I) Immunohistochemical staining of paraffin-embedded lung tissue sections at 7 and 56 days showed CD68, MMP9, and MMP12 expression. Nuclei were stained by hematoxylin (blue). (J–L) Identification of MMP9 and MMP12 protein levels in mouse lung tissues at 7 and 56 days by western blot. The levels of MMP9 and MMP12 were normalized to those of β-actin. (M) Representative images for the immunohistochemical staining of collagen I in paraffin-embedded lung tissue sections 56 days after CS instillation. Nuclei were stained by hematoxylin (blue). (I,M) Scale bar, 50 µm. Experiments were performed three times. Data are shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Mouse Assay, Inhibition, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Expressing

    Expression of 4-1BB (CD137) and 4-1BBL (CD137L) on pulmonary macrophages. After 7-days exposure to crystalline silica, mice were sacrificed. The lungs were prepared as single-cell suspensions for flow cytometric analyses ( n = 3–4). (A) Representative plots of flow cytometric analyses for 4-1BB and 4-1BBL on alveolar macrophages (AMs) and interstitial macrophages (IMs). (B,C) The percentage of AMs expressing 4-1BB and 4-1BBL. (D,E) The frequency of AMs and IMs in CD45+ cells from the lungs. (F,G) The percentage of IMs expressing 4-1BB and 4-1BBL. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Expression of 4-1BB (CD137) and 4-1BBL (CD137L) on pulmonary macrophages. After 7-days exposure to crystalline silica, mice were sacrificed. The lungs were prepared as single-cell suspensions for flow cytometric analyses ( n = 3–4). (A) Representative plots of flow cytometric analyses for 4-1BB and 4-1BBL on alveolar macrophages (AMs) and interstitial macrophages (IMs). (B,C) The percentage of AMs expressing 4-1BB and 4-1BBL. (D,E) The frequency of AMs and IMs in CD45+ cells from the lungs. (F,G) The percentage of IMs expressing 4-1BB and 4-1BBL. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Expressing, Mouse Assay, Affinity Magnetic Separation

    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections

    doi: 10.1016/j.omtm.2017.09.003

    Figure Lengend Snippet: Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p

    Article Snippet: ELISA plates coated with 200 ng of H1N1 (A/WSN/33) HA, 50 ng of H3N2 (A/Aichi/2/1968) HA1 or HA subunit, or 200 ng of H10N8 (A/Jiangxi-Donghu/346/2013) HA (Sino Biologicals, Beijing, China) were blocked and incubated with serially diluted mouse serum.

    Techniques: Mouse Assay

    Neutralizing titers for SARS-CoV-2 and SARS-CoV in COVID-19 subject plasma. a Neutralization assay with S-RBD-specific NAb, healthy control plasma, and a COVID-19 patient plasma. Threefold serial dilutions of NAb from 10 μg/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with spike protein pseudovirus and added to 293-ACE2 cells. GFP expression was analyzed by flow cytometry 3 days post infection. b SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as an outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups ( n = 113). c NT50 of COVID-19 patient and plasma donor groups subdivided into males and females ( n = 113). d Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups ( n = 104), 293-ACE2 cells were infected and RFP expression was determined at day 3 using flow cytometry. e Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV ( n = 46). Two-tailed Mann–Whitney U test was used to determine the statistical significances in figures ( b ), ( c ) and ( d ) and two-tailed Spearman’s was used for figure ( e ). Horizontal bars in ( b ), ( c ) and ( d ) indicate mean values.

    Journal: Communications Biology

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

    doi: 10.1038/s42003-021-01649-6

    Figure Lengend Snippet: Neutralizing titers for SARS-CoV-2 and SARS-CoV in COVID-19 subject plasma. a Neutralization assay with S-RBD-specific NAb, healthy control plasma, and a COVID-19 patient plasma. Threefold serial dilutions of NAb from 10 μg/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with spike protein pseudovirus and added to 293-ACE2 cells. GFP expression was analyzed by flow cytometry 3 days post infection. b SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as an outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups ( n = 113). c NT50 of COVID-19 patient and plasma donor groups subdivided into males and females ( n = 113). d Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups ( n = 104), 293-ACE2 cells were infected and RFP expression was determined at day 3 using flow cytometry. e Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV ( n = 46). Two-tailed Mann–Whitney U test was used to determine the statistical significances in figures ( b ), ( c ) and ( d ) and two-tailed Spearman’s was used for figure ( e ). Horizontal bars in ( b ), ( c ) and ( d ) indicate mean values.

    Article Snippet: Pseudotyped lentivirus production and titer measurement Lentivector plasmids containing RFP or GFP reporter genes were co-transfected with either SARS-CoV-2 spike protein or SARS -CoV spike protein (Human SARS coronavirus (SARS-CoV) spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized) from SinoBiological) plasmids into HEK-293T cells using Lipofectamine TM 3000 (Invitrogen) according to the manufacturer’s protocol.

    Techniques: Neutralization, Incubation, Expressing, Flow Cytometry, Infection, Two Tailed Test, MANN-WHITNEY

    Neutralization of SARS-CoV-2 and SARS-CoV pseudoviruses with soluble ACE2 and Nabs. a Illustration of spike-protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. b SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 h and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD-specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-NAb) or soluble ACE2 (sACE2) proteins for 1 h at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Red, green, blue and turquoise colored lines show SARS-CoV-2 S-RBD NAb#1, SARS-CoV-2 S-RBD non-NAb, soluble ACE2 #1 and #2, respectively. d Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and two different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Red lines represent antibodies while blue lines show soluble ACE2 molecules. Dot, triangle, square, asterisk, circle and star symbols indicate SARS-CoV-2 S-RBD NAb #1, #2, #3, #4, soluble ACE2 #1 and #2, respectively. Graphs in ( c ) and ( d ) represent three replicates of the experiments. Error bars indicate one standard deviation of mean values.

    Journal: Communications Biology

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

    doi: 10.1038/s42003-021-01649-6

    Figure Lengend Snippet: Neutralization of SARS-CoV-2 and SARS-CoV pseudoviruses with soluble ACE2 and Nabs. a Illustration of spike-protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. b SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 h and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD-specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-NAb) or soluble ACE2 (sACE2) proteins for 1 h at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Red, green, blue and turquoise colored lines show SARS-CoV-2 S-RBD NAb#1, SARS-CoV-2 S-RBD non-NAb, soluble ACE2 #1 and #2, respectively. d Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and two different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Red lines represent antibodies while blue lines show soluble ACE2 molecules. Dot, triangle, square, asterisk, circle and star symbols indicate SARS-CoV-2 S-RBD NAb #1, #2, #3, #4, soluble ACE2 #1 and #2, respectively. Graphs in ( c ) and ( d ) represent three replicates of the experiments. Error bars indicate one standard deviation of mean values.

    Article Snippet: Pseudotyped lentivirus production and titer measurement Lentivector plasmids containing RFP or GFP reporter genes were co-transfected with either SARS-CoV-2 spike protein or SARS -CoV spike protein (Human SARS coronavirus (SARS-CoV) spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized) from SinoBiological) plasmids into HEK-293T cells using Lipofectamine TM 3000 (Invitrogen) according to the manufacturer’s protocol.

    Techniques: Neutralization, Incubation, Expressing, Infection, Blocking Assay, Standard Deviation

    Development of SARS-CoV-2 and SARS-CoV spike-protein pseudotyped lentiviruses. a Schematic illustration of spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b 293 cells transfected with spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c Schematic representation of spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate spike pseudovirus that in turn infect engineered cells over-expressing wild-type ACE2 or ACE2-mKO2 fusion. d Infection of wild-type 293 cells with either bald lentiviruses generated without envelope plasmid or spike protein pseudovirus. e Infection of 293-ACE2 cells with bald and spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f The titrations of SARS-CoV-2 and SARS-CoV spike protein pseudoviruses encoding RFP. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Brown, red, salmon and orange-colored lines show direct infection, first, second and third freeze/thaw cycles, respectively. ACE2-IRES-GFP expressing 293 cells were incubated with threefold serial dilutions of virus supernatant, stored for several hours at 4 °C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e., ACE2+). Titration experiments were replicated twice except for the ‘1 freeze/thaw cycle’ for which titrations were replicated four times. Error bars represent 1 standard deviation of mean values.

    Journal: Communications Biology

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

    doi: 10.1038/s42003-021-01649-6

    Figure Lengend Snippet: Development of SARS-CoV-2 and SARS-CoV spike-protein pseudotyped lentiviruses. a Schematic illustration of spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b 293 cells transfected with spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c Schematic representation of spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate spike pseudovirus that in turn infect engineered cells over-expressing wild-type ACE2 or ACE2-mKO2 fusion. d Infection of wild-type 293 cells with either bald lentiviruses generated without envelope plasmid or spike protein pseudovirus. e Infection of 293-ACE2 cells with bald and spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f The titrations of SARS-CoV-2 and SARS-CoV spike protein pseudoviruses encoding RFP. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Brown, red, salmon and orange-colored lines show direct infection, first, second and third freeze/thaw cycles, respectively. ACE2-IRES-GFP expressing 293 cells were incubated with threefold serial dilutions of virus supernatant, stored for several hours at 4 °C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e., ACE2+). Titration experiments were replicated twice except for the ‘1 freeze/thaw cycle’ for which titrations were replicated four times. Error bars represent 1 standard deviation of mean values.

    Article Snippet: Pseudotyped lentivirus production and titer measurement Lentivector plasmids containing RFP or GFP reporter genes were co-transfected with either SARS-CoV-2 spike protein or SARS -CoV spike protein (Human SARS coronavirus (SARS-CoV) spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized) from SinoBiological) plasmids into HEK-293T cells using Lipofectamine TM 3000 (Invitrogen) according to the manufacturer’s protocol.

    Techniques: Expressing, Staining, Transfection, Negative Control, Flow Cytometry, Infection, Plasmid Preparation, Generated, Incubation, Titration, Standard Deviation

    SARS-CoV-2 nucleoprotein was present in several tissues of COVID-19-negative donors. ( a ) Representative images of donor tissue specimens analyzed by Western blot. Labels in the images show the location of tissue sampling in each organ/tissue. ( b ) Western blot analysis for SARS-CoV-2 nucleoprotein on total protein extracts from tissues of 8 different deceased donor PVW specimens, RV specimens from healthy donors (collected prior to COVID-19 pandemic), and LV specimen from COVID-19-positive deceased patient. GAPDH was used as a loading control protein. ( c , d ) Western blot analysis for SARS-CoV-2 nucleoprotein on total protein extracts from different cardiac and valve tissues of 2 deceased donors (#20-22 and #20-24) positive for SARS-CoV-2 serological assay. RV specimen from a healthy donor (collected prior to COVID-19 pandemic) and LV specimen from a COVID-19-positive deceased patient were used as controls. GAPDH was used as the loading control protein. PVW: pulmonary vein wall; RA: right atrium; IAS: interatrial septum; LA: left atrium; RV: right ventricle; IVS: interventricular septum; LV: left ventricle; AW: aortic wall; AVL: aortic valve leaflet.

    Journal: Diagnostics

    Article Title: Presence of SARS-CoV-2 Nucleoprotein in Cardiac Tissues of Donors with Negative COVID-19 Molecular Tests

    doi: 10.3390/diagnostics11040731

    Figure Lengend Snippet: SARS-CoV-2 nucleoprotein was present in several tissues of COVID-19-negative donors. ( a ) Representative images of donor tissue specimens analyzed by Western blot. Labels in the images show the location of tissue sampling in each organ/tissue. ( b ) Western blot analysis for SARS-CoV-2 nucleoprotein on total protein extracts from tissues of 8 different deceased donor PVW specimens, RV specimens from healthy donors (collected prior to COVID-19 pandemic), and LV specimen from COVID-19-positive deceased patient. GAPDH was used as a loading control protein. ( c , d ) Western blot analysis for SARS-CoV-2 nucleoprotein on total protein extracts from different cardiac and valve tissues of 2 deceased donors (#20-22 and #20-24) positive for SARS-CoV-2 serological assay. RV specimen from a healthy donor (collected prior to COVID-19 pandemic) and LV specimen from a COVID-19-positive deceased patient were used as controls. GAPDH was used as the loading control protein. PVW: pulmonary vein wall; RA: right atrium; IAS: interatrial septum; LA: left atrium; RV: right ventricle; IVS: interventricular septum; LV: left ventricle; AW: aortic wall; AVL: aortic valve leaflet.

    Article Snippet: The in-depth analysis in cardiac specimens of donors #20-22 and #20-24 further highlighted the presence of SARS-CoV-2 nucleoprotein in other heart regions, especially in the right atrium and right ventricle (RA and RV).

    Techniques: Western Blot, Sampling, Serologic Assay