streptavidin conjugated alkaline phosphatase  (Thermo Fisher)


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    Name:
    Streptavidin Alkaline Phosphatase Conjugate
    Description:
    The streptavidin alkaline phosphatase conjugate can be used to detect biotin in a signal amplification scheme in conjunction with chromogenic substrates or fluorogenic substrates
    Catalog Number:
    s921
    Price:
    None
    Applications:
    Antibodies and Secondary Detection|Cell Analysis
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher streptavidin conjugated alkaline phosphatase
    INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after <t>streptavidin-gold</t> labeling ( e ) or Ni-NTA-gold labeling ( f ).
    The streptavidin alkaline phosphatase conjugate can be used to detect biotin in a signal amplification scheme in conjunction with chromogenic substrates or fluorogenic substrates
    https://www.bioz.com/result/streptavidin conjugated alkaline phosphatase/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated alkaline phosphatase - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism"

    Article Title: Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.3199

    INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after streptavidin-gold labeling ( e ) or Ni-NTA-gold labeling ( f ).
    Figure Legend Snippet: INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after streptavidin-gold labeling ( e ) or Ni-NTA-gold labeling ( f ).

    Techniques Used: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis, Staining, Labeling

    ICEBERG interacts with caspase-1 CARD by co-mixing. ( a ) Co-expression of His-GFP-caspase-1 CARD and biotinylated ICEBERG. Streptavidin-AP and anti-6×His-tag Western blots were shown to confirm the presence of ICEBERG in complex with His-GFP-caspase-1 CARD after Ni-NTA affinity chromatography. L: Whole cell lysate. S: Soluble fraction. U: Ni-NTA unbound. E: Ni-NTA elute. ( b ) The complex eluted from the void of a Superdex 200 column. ( c–d ) Negative-stain electron micrographs of the complex (as shown in panel b at 7 ml) between His-GFP-caspase-1 CARD and biotinylated ICEBERG after Ni-NTA-gold labeling ( c ) or streptavidin-gold labeling ( d ).
    Figure Legend Snippet: ICEBERG interacts with caspase-1 CARD by co-mixing. ( a ) Co-expression of His-GFP-caspase-1 CARD and biotinylated ICEBERG. Streptavidin-AP and anti-6×His-tag Western blots were shown to confirm the presence of ICEBERG in complex with His-GFP-caspase-1 CARD after Ni-NTA affinity chromatography. L: Whole cell lysate. S: Soluble fraction. U: Ni-NTA unbound. E: Ni-NTA elute. ( b ) The complex eluted from the void of a Superdex 200 column. ( c–d ) Negative-stain electron micrographs of the complex (as shown in panel b at 7 ml) between His-GFP-caspase-1 CARD and biotinylated ICEBERG after Ni-NTA-gold labeling ( c ) or streptavidin-gold labeling ( d ).

    Techniques Used: Expressing, Western Blot, Affinity Chromatography, Staining, Labeling

    2) Product Images from "Colorimetric Focus-Forming Assay with Automated Focus Counting by Image Analysis for Quantification of Infectious Hepatitis C Virions"

    Article Title: Colorimetric Focus-Forming Assay with Automated Focus Counting by Image Analysis for Quantification of Infectious Hepatitis C Virions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043960

    Comparison of colorimetric focus-forming assays using various secondary antibodies and chromogenic substrates. (A) Huh-7.5 cells inoculated with serial dilutions of HCV were immunostained with monoclonal anti-HCV core antibody and secondary antibodies conjugated with different enzymes, as indicated, followed by chromogenic development using various substrates, and image scanning by ELISpot reader. (B) A magnified view of the scanned image of the colorimetric focus-forming assay revealed that alkaline phosphatase-conjugated secondary antibody with BCIP/NBT yielded the best results, considering background and distinctness of the foci. Biotin-2°Ab, biotin-conjugated secondary antibody; SA-AP, streptavidin-conjugated alkaline phosphatase; AP-2°Ab, alkaline phosphatase-conjugated secondary antibody; HRP-2°Ab, horseradish peroxidase-conjugated secondary antibody; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; DAB, 3,3′-diaminobenzidine; TMB, 3,3′,5,5′-tetramethylbenzidine.
    Figure Legend Snippet: Comparison of colorimetric focus-forming assays using various secondary antibodies and chromogenic substrates. (A) Huh-7.5 cells inoculated with serial dilutions of HCV were immunostained with monoclonal anti-HCV core antibody and secondary antibodies conjugated with different enzymes, as indicated, followed by chromogenic development using various substrates, and image scanning by ELISpot reader. (B) A magnified view of the scanned image of the colorimetric focus-forming assay revealed that alkaline phosphatase-conjugated secondary antibody with BCIP/NBT yielded the best results, considering background and distinctness of the foci. Biotin-2°Ab, biotin-conjugated secondary antibody; SA-AP, streptavidin-conjugated alkaline phosphatase; AP-2°Ab, alkaline phosphatase-conjugated secondary antibody; HRP-2°Ab, horseradish peroxidase-conjugated secondary antibody; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; DAB, 3,3′-diaminobenzidine; TMB, 3,3′,5,5′-tetramethylbenzidine.

    Techniques Used: Enzyme-linked Immunospot, Focus Forming Assay

    Microscopic images of HCV foci, developed using various secondary antibodies and chromogenic substrates. (A) Biotin-conjugated secondary antibody in conjunction with streptavidin-conjugated alkaline phosphatase and BCIP/NBT. (B) Alkaline phosphatase-conjugated secondary antibody and BCIP/NBT. (C) Horseradish peroxidase-conjugated secondary antibody and DAB. (D) Horseradish peroxidase-conjugated secondary antibody and DAB. (E) Fluorescence-conjugated secondary antibody. Magnification, 100×.
    Figure Legend Snippet: Microscopic images of HCV foci, developed using various secondary antibodies and chromogenic substrates. (A) Biotin-conjugated secondary antibody in conjunction with streptavidin-conjugated alkaline phosphatase and BCIP/NBT. (B) Alkaline phosphatase-conjugated secondary antibody and BCIP/NBT. (C) Horseradish peroxidase-conjugated secondary antibody and DAB. (D) Horseradish peroxidase-conjugated secondary antibody and DAB. (E) Fluorescence-conjugated secondary antibody. Magnification, 100×.

    Techniques Used: Fluorescence

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: PPARα Activators Inhibit Cytokine-Induced Vascular Cell Adhesion Molecule-1 Expression in Human Endothelial Cells
    Article Snippet: .. Enzyme immunoassay (EIA) was performed by incubating EC monolayers first with specific monoclonal antibodies against VCAM-1 (E1/6), ICAM-1 (HU5/3), or E-selectin (H18/7), then with biotinylated goat anti-mouse IgG (Vector Laboratories), and finally with streptavidin–alkaline phosphatase (Zymed Laboratories). (All monoclonal antibodies were a generous gift from Dr Michael Gimbrone, Brigham and Women’s Hospital, Boston, Mass). .. Cells were washed in PBS/1% BSA after each incubation step, and the integrity of the cellular monolayer was ensured by phase-contrast microscopy.

    Western Blot:

    Article Title: Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism
    Article Snippet: .. The presence of biotin was confirmed by Western blot using streptavidin-conjugated alkaline phosphatase (streptavidine-AP, Molecular Probes Cat# S921). ..

    Article Title: Connexin43 carboxyl-terminal peptides reduce scar progenitor and promote regenerative healing following skin wounding
    Article Snippet: .. Skin samples were collected at 2 and 6 h postwounding to determine peptide levels in the samples using anti-Cx43 (stock no. C6219, Sigma, MO, USA) immunoblots, streptavidin alkaline phosphatase (AP; stock no. S921, Molecular Probes, OR, USA) blotting and fluor-conjugated streptavidin microscopy as previously detailed in Hunter et al. [ ]. ..

    Incubation:

    Article Title: Immunogenicity and Protective Efficacy of Seasonal Human Live Attenuated Cold-Adapted Influenza Virus Vaccine in Pigs
    Article Snippet: .. Plates were incubated for 2 h at room temperature, washed five times and streptavidin alkaline phosphatase (Invitrogen, UK) was added for a further 1 h at room temperature. .. Spots were visualized using alkaline phosphatase substrate kit (Bio-Rad, UK) and the reaction was stopped using tap water.

    Article Title: Fc receptor–like 1 intrinsically recruits c-Abl to enhance B cell activation and function
    Article Snippet: .. After washing, the plates were incubated with streptavidin–alkaline phosphatase (S921, Invitrogen). .. BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) (TA-500-ABS, Thermo Fisher Scientific) served as the substrate of alkaline phosphatase.

    Article Title: HLA class II polymorphism influences the immune response to protective antigen and susceptibility to Bacillus anthracis
    Article Snippet: .. The plate contents were then discarded and plates were washed with PBS-Tween 20 (0.1%) and incubated with biotinylated anti-INFγ, then washed again before streptavidin-alkaline-phosphatase conjugate was added. ..

    Microscopy:

    Article Title: Connexin43 carboxyl-terminal peptides reduce scar progenitor and promote regenerative healing following skin wounding
    Article Snippet: .. Skin samples were collected at 2 and 6 h postwounding to determine peptide levels in the samples using anti-Cx43 (stock no. C6219, Sigma, MO, USA) immunoblots, streptavidin alkaline phosphatase (AP; stock no. S921, Molecular Probes, OR, USA) blotting and fluor-conjugated streptavidin microscopy as previously detailed in Hunter et al. [ ]. ..

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    Thermo Fisher streptavidin conjugated to alkaline phosphatase
    Streptavidin Conjugated To Alkaline Phosphatase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated to alkaline phosphatase/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated to alkaline phosphatase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Thermo Fisher streptavidin conjugated alkaline phosphatase sav alp
    Schematic illustration of tdEV sensing using a sandwich immunoassay and redox cycling on nIDEs resulting in a two-level selectivity and a two-level amplification. tdEVs are captured using C-AE tethered to electrodes (first level of selectivity). The binding of R-AE to the tdEVs completes the antibody–antigen-antibody sandwich (second-level selectivity), after which the enzyme <t>ALP</t> is introduced using a <t>biotin–SAV</t> interaction. ALP provides an enzymatic amplification of pAPP to pAP by substrate cleavage (first-level amplification), which is followed by an electrochemical signal amplification via the oxidation of pAP to pQI and subsequent redox cycling thereof between the nIDE electrodes (second-level amplification).
    Streptavidin Conjugated Alkaline Phosphatase Sav Alp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated alkaline phosphatase sav alp/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
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    84
    prozyme alkaline phosphatase conjugated streptavidin sa ap
    Detection of PD-1 and PD-L1 expression on canine cell lines. (A) PD-1 expression on CLGL-90 cells was detected with JC053 hybridoma culture supernatant. Biotinylated anti-mouse IgA and FITC-conjugated <t>streptavidin</t> was used. Mouse IgA isotype control was used as negative control. Light gray is for isotype control and dark gray is for JC053. (B) Four purified anti-PD-L1s were compared in their capacity to bind activated DH82 cells. DH82 cells were cultivated either with or without 10ng/ml IFN-γ and JC071, JC173, JC194, and JC205 binding to PD-L1 was assessed. Histograms show unstimulated DH82 without anti-PD-L1 (light gray), unstimulated DH82 with anti-PD-L1 (medium gray), and IFN-γ stimulated DH82 with anti-PD-L1 (dark gray), respectively. FITC conjugated Anti-Mouse IgG was used with all samples including no anti-PD-L1 sample, as secondary antibody. PD-1 or PD-L1 positive population was selected following FSC, SSC gating, single cell gating, and live cell gating.
    Alkaline Phosphatase Conjugated Streptavidin Sa Ap, supplied by prozyme, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alkaline phosphatase conjugated streptavidin sa ap/product/prozyme
    Average 84 stars, based on 1 article reviews
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    Image Search Results


    Schematic illustration of tdEV sensing using a sandwich immunoassay and redox cycling on nIDEs resulting in a two-level selectivity and a two-level amplification. tdEVs are captured using C-AE tethered to electrodes (first level of selectivity). The binding of R-AE to the tdEVs completes the antibody–antigen-antibody sandwich (second-level selectivity), after which the enzyme ALP is introduced using a biotin–SAV interaction. ALP provides an enzymatic amplification of pAPP to pAP by substrate cleavage (first-level amplification), which is followed by an electrochemical signal amplification via the oxidation of pAP to pQI and subsequent redox cycling thereof between the nIDE electrodes (second-level amplification).

    Journal: Nano Letters

    Article Title: Electrochemical Detection of Tumor-Derived Extracellular Vesicles on Nanointerdigitated Electrodes

    doi: 10.1021/acs.nanolett.9b02741

    Figure Lengend Snippet: Schematic illustration of tdEV sensing using a sandwich immunoassay and redox cycling on nIDEs resulting in a two-level selectivity and a two-level amplification. tdEVs are captured using C-AE tethered to electrodes (first level of selectivity). The binding of R-AE to the tdEVs completes the antibody–antigen-antibody sandwich (second-level selectivity), after which the enzyme ALP is introduced using a biotin–SAV interaction. ALP provides an enzymatic amplification of pAPP to pAP by substrate cleavage (first-level amplification), which is followed by an electrochemical signal amplification via the oxidation of pAP to pQI and subsequent redox cycling thereof between the nIDE electrodes (second-level amplification).

    Article Snippet: Streptavidin-conjugated alkaline phosphatase (SAV-ALP) was purchased from Thermo Fisher (Eindhoven, The Netherlands).

    Techniques: Amplification, Binding Assay

    Detection of PD-1 and PD-L1 expression on canine cell lines. (A) PD-1 expression on CLGL-90 cells was detected with JC053 hybridoma culture supernatant. Biotinylated anti-mouse IgA and FITC-conjugated streptavidin was used. Mouse IgA isotype control was used as negative control. Light gray is for isotype control and dark gray is for JC053. (B) Four purified anti-PD-L1s were compared in their capacity to bind activated DH82 cells. DH82 cells were cultivated either with or without 10ng/ml IFN-γ and JC071, JC173, JC194, and JC205 binding to PD-L1 was assessed. Histograms show unstimulated DH82 without anti-PD-L1 (light gray), unstimulated DH82 with anti-PD-L1 (medium gray), and IFN-γ stimulated DH82 with anti-PD-L1 (dark gray), respectively. FITC conjugated Anti-Mouse IgG was used with all samples including no anti-PD-L1 sample, as secondary antibody. PD-1 or PD-L1 positive population was selected following FSC, SSC gating, single cell gating, and live cell gating.

    Journal: PLoS ONE

    Article Title: Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function

    doi: 10.1371/journal.pone.0235518

    Figure Lengend Snippet: Detection of PD-1 and PD-L1 expression on canine cell lines. (A) PD-1 expression on CLGL-90 cells was detected with JC053 hybridoma culture supernatant. Biotinylated anti-mouse IgA and FITC-conjugated streptavidin was used. Mouse IgA isotype control was used as negative control. Light gray is for isotype control and dark gray is for JC053. (B) Four purified anti-PD-L1s were compared in their capacity to bind activated DH82 cells. DH82 cells were cultivated either with or without 10ng/ml IFN-γ and JC071, JC173, JC194, and JC205 binding to PD-L1 was assessed. Histograms show unstimulated DH82 without anti-PD-L1 (light gray), unstimulated DH82 with anti-PD-L1 (medium gray), and IFN-γ stimulated DH82 with anti-PD-L1 (dark gray), respectively. FITC conjugated Anti-Mouse IgG was used with all samples including no anti-PD-L1 sample, as secondary antibody. PD-1 or PD-L1 positive population was selected following FSC, SSC gating, single cell gating, and live cell gating.

    Article Snippet: The transferred membrane was blocked with 5% skim milk, incubated with alkaline phosphatase conjugated streptavidin (SA-AP), and developed using NBT/BCIP (Thermo Fisher Scientific) as a substrate.

    Techniques: Expressing, Negative Control, Purification, Binding Assay

    Evaluation of blocking activity by anti-canine PD-1 and PD-L1 antibodies. Antibodies were screened for their opposite ligand blocking capability. Hybridoma supernatants were added to ELISA plates coated with unbiotinylated PD-1Ig (A) or PD-L1Ig (B). After washing, biotinylated PD-L1Ig (A) or PD-1Ig (B) was used to detect blocking capacity of anti-PD-1 (A) and anti-PD-L1 (B) using HRP conjugated streptavidin to detect unblocked protein. All samples were assayed in duplicates. Each value is presented as mean ± standard deviation. In (A), JC053 value was significantly different from the rest of anti-PD-1 hybridomas with P value of 0.0002. In (B), JC071, JC173, JC194, and JC205 values were significantly different from PBS and medium values with P values less than 0.0001.

    Journal: PLoS ONE

    Article Title: Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function

    doi: 10.1371/journal.pone.0235518

    Figure Lengend Snippet: Evaluation of blocking activity by anti-canine PD-1 and PD-L1 antibodies. Antibodies were screened for their opposite ligand blocking capability. Hybridoma supernatants were added to ELISA plates coated with unbiotinylated PD-1Ig (A) or PD-L1Ig (B). After washing, biotinylated PD-L1Ig (A) or PD-1Ig (B) was used to detect blocking capacity of anti-PD-1 (A) and anti-PD-L1 (B) using HRP conjugated streptavidin to detect unblocked protein. All samples were assayed in duplicates. Each value is presented as mean ± standard deviation. In (A), JC053 value was significantly different from the rest of anti-PD-1 hybridomas with P value of 0.0002. In (B), JC071, JC173, JC194, and JC205 values were significantly different from PBS and medium values with P values less than 0.0001.

    Article Snippet: The transferred membrane was blocked with 5% skim milk, incubated with alkaline phosphatase conjugated streptavidin (SA-AP), and developed using NBT/BCIP (Thermo Fisher Scientific) as a substrate.

    Techniques: Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Expression of recombinant PD-1Ig and PD-L1Ig in D . melanogaster S2 cells. Expression of monomers was tested by Western blot. Biotinylated PD-1Ig and PD-L1Ig were expressed and secreted from D . melanogaster S2 cells. S2 cell culture supernatant expressing PD-1Ig (lane 2) or PD-L1Ig (lane 3) was analyzed against vector only control (lane 1) on Western blot in reducing condition. Alkaline phosphatase conjugated streptavidin was used to detect proteins.

    Journal: PLoS ONE

    Article Title: Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function

    doi: 10.1371/journal.pone.0235518

    Figure Lengend Snippet: Expression of recombinant PD-1Ig and PD-L1Ig in D . melanogaster S2 cells. Expression of monomers was tested by Western blot. Biotinylated PD-1Ig and PD-L1Ig were expressed and secreted from D . melanogaster S2 cells. S2 cell culture supernatant expressing PD-1Ig (lane 2) or PD-L1Ig (lane 3) was analyzed against vector only control (lane 1) on Western blot in reducing condition. Alkaline phosphatase conjugated streptavidin was used to detect proteins.

    Article Snippet: The transferred membrane was blocked with 5% skim milk, incubated with alkaline phosphatase conjugated streptavidin (SA-AP), and developed using NBT/BCIP (Thermo Fisher Scientific) as a substrate.

    Techniques: Expressing, Recombinant, Western Blot, Cell Culture, Plasmid Preparation

    Detection of PD-1 and PD-L1 expression on CHO cells expressing canine markers. Antibody specificity toward PD-1 and PD-L1 by anti-PD-1 (JC053) and anti-PD-L1s (JC071, JC173, JC194, and JC205) was tested on CHO cells by flow cytometry. (A) Anti-PD-1 and anti-PD-L1 bind to CHO-PD1 and CHO-PDL1, respectively, while they do not bind to untransfected CHO-K1. PD-1 positive population were selected following FSC, SSC gating, single cell gating, and live cell gating. PD-L1 positive population was selected following FSC, SSC gating, and single cell gating. (B) JC053, an anti-PD-1 antibody, was compared to a mouse IgA isotype control for staining CHO-PD1. Biotinylated anti-mouse IgA and APC conjugated streptavidin was used following primary antibody staining. A population of CHO-PD1 cells was selected on FSC and SSC gating. Single cells were selected and dead cells were excluded. Then, PD-1 positive cells are shown on histograms. Anti-PD-L1s were conjugated with PE and used to stain CHO-PDL1 cells. Appropriate isotype controls were included and anti-mouse IgG1 and anti-mouse IgG2a were used in combination with PE conjugated streptavidin. PE positive population is shown on histograms following FSC/SSC gating, single cell selection, and exclusion of dead cells.

    Journal: PLoS ONE

    Article Title: Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function

    doi: 10.1371/journal.pone.0235518

    Figure Lengend Snippet: Detection of PD-1 and PD-L1 expression on CHO cells expressing canine markers. Antibody specificity toward PD-1 and PD-L1 by anti-PD-1 (JC053) and anti-PD-L1s (JC071, JC173, JC194, and JC205) was tested on CHO cells by flow cytometry. (A) Anti-PD-1 and anti-PD-L1 bind to CHO-PD1 and CHO-PDL1, respectively, while they do not bind to untransfected CHO-K1. PD-1 positive population were selected following FSC, SSC gating, single cell gating, and live cell gating. PD-L1 positive population was selected following FSC, SSC gating, and single cell gating. (B) JC053, an anti-PD-1 antibody, was compared to a mouse IgA isotype control for staining CHO-PD1. Biotinylated anti-mouse IgA and APC conjugated streptavidin was used following primary antibody staining. A population of CHO-PD1 cells was selected on FSC and SSC gating. Single cells were selected and dead cells were excluded. Then, PD-1 positive cells are shown on histograms. Anti-PD-L1s were conjugated with PE and used to stain CHO-PDL1 cells. Appropriate isotype controls were included and anti-mouse IgG1 and anti-mouse IgG2a were used in combination with PE conjugated streptavidin. PE positive population is shown on histograms following FSC/SSC gating, single cell selection, and exclusion of dead cells.

    Article Snippet: The transferred membrane was blocked with 5% skim milk, incubated with alkaline phosphatase conjugated streptavidin (SA-AP), and developed using NBT/BCIP (Thermo Fisher Scientific) as a substrate.

    Techniques: Expressing, Flow Cytometry, Staining, Selection