FFWO assays were performed by first allowing viral adsorption to BHK-21 cells (MOI 25) for 1 h at 4ºC. Unbound virus was removed by washing with chilled PBS. Diluted mAbs (50 μg/ml) were added to virus adsorbed cells for 30 min at 4ºC. Cells were washed with chilled PBS. FFWO was induced by pulsing with fusion medium (RPMI 1640, 10 mM HEPES, 0.2% BSA, and 30 mM succinic acid, pH 5.5) for 2 min at 37ºC. A non-fusion control was included using control media (RPMI 1640, 10 mM HEPES, 0.2% BSA, pH 7.6). After the 37ºC pulse, cells were washed twice with chilled PBS and incubated in DMEM supplemented with 5% FBS, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM NH4Cl to prevent infection via endocytosis. Infection was allowed to proceed for 5 h and cells were detached and fixed with Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher). Cells were stained with human mAb EEEV-53 (L.E.W. and J.E.C, unpublished results) at 1 μg/ml in permeabilization buffer and incubated for 1 h at 4ºC. After two washes with permeabilization buffer, viral antigen was detected with Alexa Fluor 647 conjugated goat anti-human IgG (1:2000 dilution, Thermo Fisher). After two washes with permeabilization buffer, cells were resuspended in 100 μl and analyzed on a MACSQuant Analyzer (Miltenyi Biotec).
Alexa fluor 647 conjugated goat anti human igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-Fluor-647-conjugated goat anti-human IgG is a fluorescent-labeled secondary antibody used for detection and quantification of human immunoglobulin G (IgG) in various immunoassays and imaging applications. The antibody is conjugated to Alexa Fluor 647, a bright and photostable fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Macsquant analyzer, by Miltenyi Biotec (2 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Anti flag, by GenScript (1 mentions)
Caliber, by BD (1 mentions)
Genepix 4200a, by Molecular Devices (1 mentions)
Protoarray human protein microarrays v5, by Thermo Fisher Scientific (1 mentions)
Quadriperm dishes, by Greiner (1 mentions)
Axon 4200al, by Molecular Devices (1 mentions)
Genepix pro software, by Molecular Devices (1 mentions)
Human insulin, by Merck Group (1 mentions)
Facs buffer, by Thermo Fisher Scientific (1 mentions)
Cellquest pro 4, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
9 protocols using alexa fluor 647 conjugated goat anti human igg
1
Antibody-Mediated Viral Fusion Inhibition
2
Quantifying 5T4 Expression in Cancer Cells
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FCM was used to determine 5T4 expression in cancer cell lines. Logarithmic phase cells were cultured in 6-well plates and collected after digestion by cell dissociation buffer (Invitrogen). Cells 5×105 were incubated with anti-5T4 antibody m603 (prepared by our group) in fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.2% fetal bovine serum [FBS]) for 1.5 h on ice. After incubation, they were washed with FACS buffer twice, and then AlexaFluor 647-labeled goat anti-human IgG (H + L) (ThermoFisher) was added into the cells for 0.5 h. To evaluate the binding capacity of conjugates, cells were mixed with 100 nM ADC conjugates and non-conjugated antibodies for 1.5 h on ice, followed by Alexa Fluor 647 conjugated goat anti-human IgG (ThermoFisher) or anti-FLAG (Genscript) for 0.5 h. All of the samples were washed 3 times and then analyzed using a BD Caliber. Data analysis was performed using Flowjo 7.6.1.
3
Antibody-Mediated Viral Fusion Inhibition
4
GABAAR Subunit Expression and Antibody Binding
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Following our established protocol (Ly et al., 2018 (link)), HEK293T cells were transiently cotransfected with plasmids encoding for rat or human α1-, β3-, and γ2-subunits of GABAAR and enhanced GFP (EGFP) or EGFP only and then stained with serial dilutions of purified mAbs. From live single cells with top 30% protein expression (evaluated by EGFP signal), the MFI of the Alexa Fluor 647–conjugated goat anti-human IgG (1:400; Life Technologies) was calculated, and nonlinear regression models were determined as above.
5
Protein Microarray for Antibody Profiling
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ProtoArray Human Protein Microarrays version 5.0 (Life Technologies), containing approximately 9,400 unique full-length human proteins, were used. The assay was performed according to the manufacturer’s instructions as we have previously described (30 (link)). Briefly, protein microarray slides were probed with rIgG pools (normalized for total IgG content) by overnight incubation at 4°C. Bound rIgG was detected with an Alexa Fluor 647-conjugated goat anti-human IgG (Life Technologies). The arrays were then scanned using a GenePix 4200A (Molecular Devices) fluorescent microarray scanner and analyzed with GenePix software. The standard score (Z-score) for binding to each antigen was determined using the Immune Response Profiling function within Prospector software (Life Technologies). The selection criteria applied for binding to be considered positive was a Z-score >3.
6
ZIKV and DENV Antibody Binding in Aedes albopictus Cells
7
Protein Microarray Profiling of Sera
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ProtoArray Human Protein Microarrays v5.0 (Invitrogen, Carlsbad, CA, USA) were purchased and used according to the manufacturer’s instructions.
After blocking for 1 h at 4 °C and washing, arrays were incubated in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany) placed on a horizontal shaker (50 rpm) for 90 min at 4 °C with individual sera diluted at 1:500 in 5 mL washing buffer (0.1% Tween 20 [v/v], 1% BSA [w/v] in PBS). After washing, binding of IgG was detected by incubation with Alexa Fluor 647–conjugated goat anti–human IgG (Invitrogen, Waltham, MA, USA) diluted 1:2000 in assay buffer for 90 min at 4 °C. The arrays were washed again and dried by centrifugation. Arrays were scanned at a 10-μm resolution on a microarray scanner (Axon 4200AL with GenePix Pro Software; Molecular Devices, Sunnyvale, CA, USA), and fluorescence was detected according to the manufacturer’s instructions. Images were saved as 16-bit TIFF files, and analysis was performed using GenePix. The median net intensity in relative fluorescence units is reported for each spot.
After blocking for 1 h at 4 °C and washing, arrays were incubated in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany) placed on a horizontal shaker (50 rpm) for 90 min at 4 °C with individual sera diluted at 1:500 in 5 mL washing buffer (0.1% Tween 20 [v/v], 1% BSA [w/v] in PBS). After washing, binding of IgG was detected by incubation with Alexa Fluor 647–conjugated goat anti–human IgG (Invitrogen, Waltham, MA, USA) diluted 1:2000 in assay buffer for 90 min at 4 °C. The arrays were washed again and dried by centrifugation. Arrays were scanned at a 10-μm resolution on a microarray scanner (Axon 4200AL with GenePix Pro Software; Molecular Devices, Sunnyvale, CA, USA), and fluorescence was detected according to the manufacturer’s instructions. Images were saved as 16-bit TIFF files, and analysis was performed using GenePix. The median net intensity in relative fluorescence units is reported for each spot.
8
Insulin Receptor Binding Assay
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For FACS, CHO-hINSR cells (2×106/ml) were washed and resuspended in phosphate buffered saline (PBS) with 0.5% fatty acid-free bovine serum albumin and 0.1% sodium azide (FACS buffer; Invitrogen, Carlsbad, CA). Cells were preincubated for 10 minutes at 4°C in the presence of 100 nM human insulin (Sigma-Aldrich). Next, increasing concentrations of XMetS were added to the cell suspension and incubated at 15°C for 120 minutes. Cells were then washed and resuspended in Alexa Fluor® 647-conjugated goat anti-human IgG (1∶200; Invitrogen, Carlsbad, CA). The cells were incubated for 30 minutes at 4°C, washed twice and analyzed on a FACScan™ flow cytometer (Becton Dickinson, San Jose, CA).
9
Quantifying HIV-1 Env Surface Expression
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Upon transient cotransfection with proviral plasmids encoding green fluorescent protein (GFP) and 90K-myc-encoding constructs, HEK293T cells were harvested after 48 h. Cells were stained with the primary anti-gp120 antibody (2G12) followed by secondary staining with the Alexa Fluor 647-conjugated goat anti-human IgG (Invitrogen) (9 (link), 29 (link)). After the surface HIV-1 Env staining, cells were fixed with paraformaldehyde (PFA) and analyzed for surface HIV-1 Env levels. Due to the disproportional expression of Env inside the cell (high) as opposed to on the cell surface (low) in provirally transfected HEK293T cells (4 (link), 9 (link)), we restricted the analysis to gate R3, which contains cells driving sufficiently high viral gene expression to yield detectable cell surface Env, and normalized these signals to the respective R2 gate. Flow cytometry analysis was performed using FACSCalibur with BD CellQuest Pro 4.0.2 software (BD Pharmingen) and FlowJo V10 software (FlowJo).
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