Titrating amounts of mAbs (100 µg mL−1 in 4-fold titrations) were added to HIV-1 env-transfected 293T cells (as described above) 2 days post-transfection and incubated for 1 h at 4 °C in 1× PBS52 (link). After washing, cells were fixed with 2% para-formaldehyde (PolySciences) for 20 min at room temperature. Cells were then washed and stained with a 1:200 dilution of PE-conjugated goat anti-human IgG (Southern Biotech, #2040-09) for 1 h at room temperature. Binding was analyzed using flow cytometry by determining mean fluorescence intensities (MFIs) of PE+-stained cell populations. FlowJo v10.4 was used for data analysis and interpretation.
Pe conjugated goat anti human igg
Manufactured by Southern Biotech
Sourced in United Kingdom
PE-conjugated goat anti-human IgG is a laboratory reagent used to detect and quantify human immunoglobulin G (IgG) in samples. The product consists of goat-derived polyclonal antibodies that are conjugated to the fluorescent dye phycoerythrin (PE). This allows for the specific labeling and visualization of human IgG in various immunoassay applications.
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Lab products found in correlation
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (1 mentions)
Brij 35, by Thermo Fisher Scientific (1 mentions)
Pe conjugated goat f ab 2 anti human igg, by Southern Biotech (1 mentions)
Hrp conjugated goat anti human igg, by Southern Biotech (1 mentions)
Facsymphony a5 flow cytometer, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
4 protocols using pe conjugated goat anti human igg
1
Quantifying mAb Binding to HIV-1 Env
2
Quantifying Influenza HA-Reactive IgG
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The levels of HA-reactive IgG were measured in plasma and
in vitro stimulated B cell culture supernatants using the mPlex-Flu assay, as previously described
43 (link). The assay panels included whole HA or the head segments of influenza group 1, group 2, B strain and chimeric HA, as listed in
Table 1 . Briefly, 25
µL of plasma dilution (1:5000) or undiluted culture supernatants were incubated with 25
µL of a panel of beads coupled with HAs at room temperature for two hours on a rotary shaker (500 rpm) in the dark. Then 150
µL of phycoerythrin (PE) conjugated goat anti-human IgG (Southern Biotech, Birmingham, Al) was added and incubated at room temperature for 2 hours on a rotary shaker (500 rpm) in the dark. After wells were washed twice with PBS (pH 7.2) containing 0.1% BSA (MP Biomedical, LLC, France) and 0.1% Brij-35 (Thermo Scientific, Waltham, MA), IgG levels were analyzed on Magpix Multiplex Reader (Luminex, Austin, TX). All samples were measured in duplicate.
in vitro stimulated B cell culture supernatants using the mPlex-Flu assay, as previously described
43 (link). The assay panels included whole HA or the head segments of influenza group 1, group 2, B strain and chimeric HA, as listed in
µL of plasma dilution (1:5000) or undiluted culture supernatants were incubated with 25
µL of a panel of beads coupled with HAs at room temperature for two hours on a rotary shaker (500 rpm) in the dark. Then 150
µL of phycoerythrin (PE) conjugated goat anti-human IgG (Southern Biotech, Birmingham, Al) was added and incubated at room temperature for 2 hours on a rotary shaker (500 rpm) in the dark. After wells were washed twice with PBS (pH 7.2) containing 0.1% BSA (MP Biomedical, LLC, France) and 0.1% Brij-35 (Thermo Scientific, Waltham, MA), IgG levels were analyzed on Magpix Multiplex Reader (Luminex, Austin, TX). All samples were measured in duplicate.
3
Antibody Staining for PD-L1 and TIM-3
4
Flow Cytometry Analysis of rIgG Binding
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Flow cytometry analysis of rIgG binding to HA-expressing K530 cell lines was performed essentially as described 31 (link). Briefly, K530 cell lines were thawed from cryopreserved aliquots and expanded in culture for ≥3 days. Pooled K530 cells were incubated at 4°C for 30 min with 0.4 μg/ml recombinant human IgGs diluted in PBS plus 2% fetal bovine serum. After washing, cells were labeled with 2 μg/ml PE-conjugated goat anti-human IgG (Southern Biotech) for 30 min at 4°C. Cells were then washed and analyzed with a BD FACSymphony A5 flow cytometer. Flow cytometry data were analyzed with FlowJo software (BD).
Flow cytometry analysis of rIgG binding to HA-expressing 293F cells was performed as follows. 293F cells were transfected using PEI with either plasmid encoding full-length HA from A/Aichi/02/1968(H3N2)(X31) or with empty vector. 36 hours post-transfection, cells were incubated at 4°C for one hour with 0.4 μg/ml recombinant human IgGs diluted in PBS plus 2% fetal bovine serum. After washing, cells were labeled with BB515 mouse anti-human IgG (BD Biosciences) for 30 min at 4°C at the manufacturer’s recommended concentration, followed by washing and fixation in 2% paraformaldehyde. Cells were analyzed with a BD LSRFortessa flow cytometer. Flow cytometry data were analyzed with FlowJo software (BD).
Flow cytometry analysis of rIgG binding to HA-expressing 293F cells was performed as follows. 293F cells were transfected using PEI with either plasmid encoding full-length HA from A/Aichi/02/1968(H3N2)(X31) or with empty vector. 36 hours post-transfection, cells were incubated at 4°C for one hour with 0.4 μg/ml recombinant human IgGs diluted in PBS plus 2% fetal bovine serum. After washing, cells were labeled with BB515 mouse anti-human IgG (BD Biosciences) for 30 min at 4°C at the manufacturer’s recommended concentration, followed by washing and fixation in 2% paraformaldehyde. Cells were analyzed with a BD LSRFortessa flow cytometer. Flow cytometry data were analyzed with FlowJo software (BD).
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