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    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp reporter genes/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gfp reporter genes - by Bioz Stars, 2021-09
    86/100 stars

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    1) Product Images from "SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus"

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

    Journal: Communications Biology

    doi: 10.1038/s42003-021-01649-6

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    Related Articles

    Produced:

    Article Title: Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody
    Article Snippet: .. SARS-CoV-2 S and SARS_CoV-2 S N501Y pseudotyped retroviral particles were produced in HEK293T cells as described previously . ..

    Article Title: Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2
    Article Snippet: .. For the generation of 293T cells stably expressing SARS-CoV-2 Spike the same technique than previously described has been used ( ): VSV-G pseudotyped lentivirus packaging the SARS-CoV-2 Spike gene was produced in 293T using a third-generation lentiviral vector system. ..

    Expressing:

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

    Article Title: Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2
    Article Snippet: .. For the generation of 293T cells stably expressing SARS-CoV-2 Spike the same technique than previously described has been used ( ): VSV-G pseudotyped lentivirus packaging the SARS-CoV-2 Spike gene was produced in 293T using a third-generation lentiviral vector system. ..

    Article Title: Semaphorin 3A attenuates cardiac autonomic disorders and reduces inducible ventricular arrhythmias in rats with experimental myocardial infarction
    Article Snippet: .. In order to knockdown sema 3A expression, 80 μl virus solution including 1.09 × 109 TU/ml lentivirus-sema 3A-shRNA encoding green fluorescent protein(GFP) and containing sema 3A shRNA (MI-SiRNA group, n = 17) was injected intramyocardially at four sites in the peri-infarcted myocardium (~2 mm around the infarcted area), as reported previously [ ]. ..

    Plasmid Preparation:

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

    Article Title: Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2
    Article Snippet: .. For the generation of 293T cells stably expressing SARS-CoV-2 Spike the same technique than previously described has been used ( ): VSV-G pseudotyped lentivirus packaging the SARS-CoV-2 Spike gene was produced in 293T using a third-generation lentiviral vector system. ..

    Stable Transfection:

    Article Title: Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2
    Article Snippet: .. For the generation of 293T cells stably expressing SARS-CoV-2 Spike the same technique than previously described has been used ( ): VSV-G pseudotyped lentivirus packaging the SARS-CoV-2 Spike gene was produced in 293T using a third-generation lentiviral vector system. ..

    Inhibition:

    Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus
    Article Snippet: .. Monoclonal antibody inhibition assay Virus (MOI:0.5) incubated with respective mAbs (2 μg ml−1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 104 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media. ..

    Incubation:

    Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus
    Article Snippet: .. Monoclonal antibody inhibition assay Virus (MOI:0.5) incubated with respective mAbs (2 μg ml−1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 104 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media. ..

    shRNA:

    Article Title: Semaphorin 3A attenuates cardiac autonomic disorders and reduces inducible ventricular arrhythmias in rats with experimental myocardial infarction
    Article Snippet: .. In order to knockdown sema 3A expression, 80 μl virus solution including 1.09 × 109 TU/ml lentivirus-sema 3A-shRNA encoding green fluorescent protein(GFP) and containing sema 3A shRNA (MI-SiRNA group, n = 17) was injected intramyocardially at four sites in the peri-infarcted myocardium (~2 mm around the infarcted area), as reported previously [ ]. ..

    Injection:

    Article Title: Semaphorin 3A attenuates cardiac autonomic disorders and reduces inducible ventricular arrhythmias in rats with experimental myocardial infarction
    Article Snippet: .. In order to knockdown sema 3A expression, 80 μl virus solution including 1.09 × 109 TU/ml lentivirus-sema 3A-shRNA encoding green fluorescent protein(GFP) and containing sema 3A shRNA (MI-SiRNA group, n = 17) was injected intramyocardially at four sites in the peri-infarcted myocardium (~2 mm around the infarcted area), as reported previously [ ]. ..

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    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp reporter genes/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gfp reporter genes - by Bioz Stars, 2021-09
    86/100 stars
      Buy from Supplier

    94
    Sino Biological monomeric red fluorescent protein mrfp green fluorescent protein gfp light chain 3
    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.
    Monomeric Red Fluorescent Protein Mrfp Green Fluorescent Protein Gfp Light Chain 3, 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/monomeric red fluorescent protein mrfp green fluorescent protein gfp light chain 3/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monomeric red fluorescent protein mrfp green fluorescent protein gfp light chain 3 - by Bioz Stars, 2021-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    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