Percentage of inhibition of RBD binding to ACE2 by plasma/serum was analyzed through a flow cytometry cell-based assay as previously described (25 (link)). This assay was validated by direct comparison of 50% inhibitory concentration (IC50) neutralization values obtained applying a pseudovirus-based neutralization assay using HIV-based pseudovirus and ACE2 expressing 293T cells (56 (link)). Briefly, a murine stable cell line expressing the human ACE2 receptor (1.2 × 103 300.19-ACE2 cells per well in a 96-well plate) was incubated with RBD-mFc fusion proteins (4 μg/mL), previously exposed to plasma/serum samples at a dilution of 1:50 in PBS for 30 min at 4°C. Cells were stained with anti-mouse IgG-PE (Jackson ImmunoResearch), washed, and analyzed by Flow cytometry using standard procedures. Samples were acquired with a FACSCanto II (BD Biosciences) and analyzed with FlowJo Xv10.0.7 (Tree Star, Inc) software (57 (link)). The surrogate neutralization assay was performed in a subset of 50 and 265 samples from the Munich and Barcelona cohorts, respectively. Samples were randomly selected covering the whole range of IgG-RBD levels at the Luminex.
Anti mouse igg pe
Manufactured by Jackson ImmunoResearch
Sourced in Cameroon
Anti-mouse IgG-PE is a conjugated secondary antibody used for detection in immunological assays. It binds to mouse immunoglobulin G (IgG) and is labeled with the fluorescent dye phycoerythrin (PE).
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Flowjox v10, by Tree Star (2 mentions)
Rabbit anti smad7 or irrelevant antibody, by Santa Cruz Biotechnology (2 mentions)
Donkey anti rabbit igg cy, by Jackson ImmunoResearch (2 mentions)
Casein protein, by Agilent Technologies (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Fitc donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Cytoperm buffer, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Goat anti mouse igg pe, by Jackson ImmunoResearch (1 mentions)
Mouse anti myc, by Bio-Rad (1 mentions)
Facs canto 2, by Jackson ImmunoResearch (1 mentions)
Cytofix, by BD (1 mentions)
Falcon tube, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Lysing buffer, by Lonza (1 mentions)
Facscanto, by BD (1 mentions)
Apc anti mouse cd49b, by BioLegend (1 mentions)
10 protocols using anti mouse igg pe
1
SARS-CoV-2 RBD-ACE2 Binding Inhibition Assay
2
SARS-CoV-2 Neutralization Assay using Flow Cytometry
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Percentage of inhibition of RBD binding to ACE2 by plasma/serum was analyzed through a flow cytometry cell-based assay as previously described (25 (link)). This assay was validated by direct comparison of 50% inhibitory concentration (IC50) neutralization values obtained applying a pseudovirus-based neutralization assay using HIV-based pseudovirus and ACE2 expressing 293T cells (56 (link)). Briefly, a murine stable cell line expressing the human ACE2 receptor (1.2 × 103 300.19-ACE2 cells per well in a 96-well plate) was incubated with RBD-mFc fusion proteins (4 μg/mL), previously exposed to plasma/serum samples at a dilution of 1:50 in PBS for 30 min at 4°C. Cells were stained with anti-mouse IgG-PE (Jackson ImmunoResearch), washed, and analyzed by Flow cytometry using standard procedures. Samples were acquired with a FACSCanto II (BD Biosciences) and analyzed with FlowJo Xv10.0.7 (Tree Star, Inc) software (57 (link)). The surrogate neutralization assay was performed in a subset of 50 and 265 samples from the Munich and Barcelona cohorts, respectively. Samples were randomly selected covering the whole range of IgG-RBD levels at the Luminex.
3
Characterizing Nanobody Cross-Reactivity to CXCR7
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Example 12
HEK293 cells transfected respectively with pcDNA3.1-hCXCR7 and pcDNA3.1-mCXCR7 were used to test cross-reactive binding of Nanobodies to mouse CXCR7 in FACS analysis. Cells were incubated with 32 nM Mab 11G8 (R&D), Mab 9C4 (MBL), Mab 8F11 (Biolegend) or with 800 nM Nanobody for 2 h at 4° C. Nanobody binding was detected using mouse anti-myc (Serotec) and anti-mouse IgG-PE (Jackson Immunoresearch) and Mab binding by goat anti-mouse IgG-PE (Jackson Immunoresearch). Nanobodies 08A10, 14G03, 07B11 and Mab9C4 are not cross-reactive to mouse CXCR7, Nanobodies 08A05 and 07C03 are partially cross-reactive with mouse CXCR7 and Mab 8F11, Mab 11G8 and 01C10 are cross-reactive with mouse CXCR7 (Table B-11).
Cross-reactive binding to cynomolgus CXCR7 was assessed in the same way. Nanobodies 08A10, 14G03, 07B11, 08A05, 07C03, 01C10 and Mab 9C4, Mab 8F11 and Mab 11G8 are all cross-reactive to cynomolgus CXCR7 (Table B-11).
4
Immunofluorescence Staining of NF-κB and Smad7
5
Binding Analysis of CXCR7 Nanobodies
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Example 8
Serial dilutions of purified proteins (concentration range: 400 nM-180 pM) were incubated with stable HEK-CXCR7 cells for 30 min at 4° C. and binding was detected using anti-mouse anti-myc (Serotec) and anti-mouse IgG-PE (Jackson Immunoresearch). The half maximal effective concentration (EC50) values and upper plateau levels of selected clones are depicted in Table B-9. These data confirm the screening data and underscore that the indicated Nanobodies bind to cellular human CXCR7.
6
Quantifying Intracellular P2RX7 Expression
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To assess the expression of P2RX7, 2x105 CM cells (WT, pcDNA3- or P2RX7-transfected) were mock- or MVA-PK1L-OVA (MOI1) infected. After 20hpi, cells were stained with fixable viability dye eFluor 660 (1:2000) for 20min on ice, permeabilized with BD Cytofix for 15min on ice and then stained with anti-P2RX7 (1:200) for one hour on ice to quantify the intracellular presence of P2RX7. Cells were further incubated with anti-mouse-IgG-PE (Jackson laboratories) (1:200) secondary antibody for 30min on ice, washed and immediately used for FACS analysis by FACS Canto II.
7
Immunofluorescence Staining of NF-κB and Smad7
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Cells were fixed and permeabilized (20 min with Cytofix/Cytoperm; BD Biosciences), washed (Cytoperm Buffer; BD Biosciences), and then blocked with casein protein (DAKO; Carpinteria, CA) for 1 h. For detection of NF-κB p65, cells were incubated with rabbit anti-NF-κB p65 and mouse anti- HCMV p63-27, specific for IE1, or irrelevant antibody (0.05 mg/mL) for 90 min (Santa Cruz Biotechnology, Santa Cruz, CA). Cells were washed and then incubated with donkey anti-rabbit IgG-FITC or anti-mouse IgG-PE (30 min, 1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). For detection of Smad7, cells were incubated with rabbit anti-Smad7 or irrelevant antibody (overnight at 4°C, 1:50; Santa Cruz Biotechnology, Dallas, TX), washed, and then incubated with donkey anti-rabbit IgG-Cy (45 min, 1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were washed with PBS and counter-stained with DAPI, and then visualized by confocal microscopy.
8
Flow Cytometric Analysis of CD21 Expression
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To assess the expression of CD21 on the cell surface, a flow cytometry assay was performed. Cells were handled in a 1.5-ml Eppendorf tube. All the procedures were done on ice. Briefly, 1×106 cells/samples were washed in PBS. For the detection of CD21, cells were incubated with primary (unconjugated) anti-CD21 antibody (1.3 μg/ml) for 20 min, washed twice with PBS, then incubated with PE-labeled secondary antibody (anti-mouse IgG-PE, Jackson Immunoresearch #115–116-068, diluted 1: 100 in PBS) for 30 min. Cells were then washed and filtered into polystyrene round-bottom 12×75 mm BD Falcon tubes (the filter was provided with the BD Falcon tube), and, finally, cells were counted on a BD FACSCalibur flow cytometer. BD CellQuest Pro software was used to analyze the cell populations (BD Biosciences).
9
Detailed Flow Cytometry Procedure for HA-Tagged and Fc Fusion Protein Detection
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Flow cytometry was performed using standard procedures [26 (link)]. To determine the cell surface expression of HA-tagged proteins, COS-7 cells were stained with the anti-HA mAb [22 (link)], followed by anti-mouse IgG-PE (Jackson ImmunoResearch, Ely, Cambridgeshire, UK). For Fc fusion protein staining, 8 µg/mL of each Fc fusion protein were used, followed by incubation with the anti-human Fc IgG mAb and by anti-mouse IgG-PE. An irrelevant Fc fusion protein (CTL-Fc) was always used as a negative control. To minimize non-specific staining, all incubations were carried out in the presence of 20% rabbit serum (Linus) and 1% fetal bovine serum in PBS. Samples were analyzed using FACSCalibur (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree star Inc, Ashland, OR, USA).
10
In Vitro IgE Quantification in Mice
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Whole blood cells from naive or peanut-sensitized mice were incubated after lysing of the erythrocytes (Lysing buffer; Lonza, Walkersville, Md) with serum of naive or CuMVtt-Ara h 1-immunized mice (1:5) together with peanut extract (1 mg/mL) in RPMI 164 for 30 minutes at room temperature. After washing, cells were stained with anti-mouse IgE-FITC (BD Becton Dickinson, Allschwil, Switzerland), anti-mouse CD49b-APC (BioLegend), and anti-mouse IgG-PE (Jackson ImmunoResearch, Cambridgeshire, United Kingdom). Measurements were performed with FACS Canto (BD Biosciences) and analysis with FlowJo software (FlowJo LCC).
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