Antigen specific antibody subclass and isotypes, and FcγR binding was analyzed by Luminex multiplexing. The antigens were coupled to magnetic Luminex beads (Luminex Corp, TX, USA) by carbodiimide-NHS ester-coupling with an individual region per antigen. Coupled beads were incubated with different plasma dilutions (1:100 for IgG2, IgG3, IgG4, IgM and IgA1, 1:500 for IgG1 and 1:1,000 for FcγR probing) for 2 hours at room temperature in 384 well plates (Greiner Bio-One, Germany). Unbound antibodies were washed away and subclasses, isotypes were detected with a respective PE-conjugated antibody (anti-human IgG1, IgG2, IgG3, IgG4, IgM or IgA1 all SouthernBiotech, AL, USA) at a 1:100 dilution. For the analysis of FcγR binding PE-Streptavidin (Agilent Technologies, CA, USA) was coupled to recombinant and biotinylated human FcγR2a, FcγR2b, FcγR3a, or FcγR3b protein. Coupled FcγR were used as a secondary probe at a 1:1000 dilution. After 1 h incubation, excessive secondary reagent was washed away and the relative antibody concentration per antigen determined on an IQue analyzer (IntelliCyt).
Ique analyzer
Manufactured by Intellicyt
Sourced in United States
The IQue analyzer is a high-throughput flow cytometry system designed to automate and streamline sample analysis. The core function of the IQue analyzer is to rapidly measure and analyze multiple characteristics of single cells or particles within a sample. This equipment provides researchers with a powerful tool for applications such as cell-based assays, immunophenotyping, and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Fluosphere neutravidin beads, by Thermo Fisher Scientific (15 mentions)
Pe streptavidin, by Agilent Technologies (10 mentions)
384 well plate, by Greiner (6 mentions)
Gvb buffer, by Boston BioProducts (5 mentions)
Anti guinea pig c3 fitc antibody, by MP Biomedicals (4 mentions)
Thp 1 monocytes, by American Type Culture Collection (3 mentions)
Pe conjugated secondary antibody for igg1, by Southern Biotech (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Monensin, by BD (2 mentions)
Maxisporp elisa plates, by Thermo Fisher Scientific (2 mentions)
Fluorescent neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Nhs sulfo lc lc kit, by Thermo Fisher Scientific (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Anti human igg1, by Southern Biotech (1 mentions)
Rosettesep nk cell enrichment kit, by STEMCELL (1 mentions)
Anti human cd107a, by BD (1 mentions)
Rhil 15, by STEMCELL (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Corning (1 mentions)
32 protocols using ique analyzer
1
Multiplexed Antibody Profiling
2
Multiplexed Antibody Profiling Assay
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Antigen-specific antibody subclasses and isotypes, as well as FcγR binding, were analyzed by Luminex multiplexing in technical replicates. The antigens were coupled to magnetic Luminex beads (Luminex Corp) by carbodiimide- N-hydroxysulfosuccinimide (Sulfo-NHS) ester-coupling with an individual region per antigen. Coupled beads were incubated with different plasma dilutions (1:100 for IgG2, IgG3, IgG4, IgM, and IgA1; 1:500 for IgG1 and 1:1,000 for FcγR probing) for 2 hours at room temperature in 384 well plates (Greiner Bio-One). Unbound antibodies were removed by washing and subclasses and isotypes were detected with a respective phycoerythrin (PE)-conjugated antibody (anti-human IgG1 (Cat# 9052-09, RRID:AB_2796621), IgG2 (Cat# 9060-09, RRID:AB_2796635), IgG3 (Cat# 9210-09, RRID:AB_2796701), IgG4 (Cat# 9200-09, RRID:AB_2796693), IgM (Cat# 9020-09, RRID:AB_2796577), or IgA1 (Cat# 9130-09, RRID:AB_2796656) all from Southern Biotech) at a 1:100 dilution. For analysis of FcγR binding PE-Streptavidin (Agilent Technologies) was coupled to recombinant and biotinylated human FcγR2a, FcγR2b, FcγR3a, or FcγR3b protein. Coupled FcγRs were used as a secondary probe at a 1:1000 dilution. After a 1 hour incubation, excessive secondary reagent was removed by washing and the relative antibody concentration per antigen was determined on an IQue analyzer (IntelliCyt).
3
Multiplex Luminex Assay for Antibody Profiling
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Antigen-specific antibody isotype, subclass, and Fc receptor binding profiles were analyzed using a custom multiplex Luminex assay38 (link). The antigens were coupled directly onto the Luminex microspheres (Luminex Corp) via carboxy coupling. Coupled beads were incubated with diluted plasma samples overnight at 4 °C. Following overnight incubation, non-specific antibodies were washed off and the immune complexes were incubated with Ig isotypes or subclasses with a 1:100 diluted PE-conjugated secondary antibody for IgG1 (clone: HP6001), IgG2 (clone: 31-7-4), IgG3 (clone: HP6050), IgG4 (clone: HP6025), IgM (clone: SA-DA4), IgA1 (clone: B3506B4), or IgA2 (clone: A9604D2) (all Southern Biotech). For avidity analysis. immune-complexes were incubated for 10 min with 2.5 M urea or PBS (no urea control) prior to PBS wash and addition of the IgG1 secondary probe. For the FcγR binding, the secondary probe is a PE-streptavidin (Agilent Technologies) coupled recombinant and biotinylated human FcγR protein. Beads with secondary antibodies were incubated on a shaker at room temperature for 1 hour. Following incubation, excessive antibodies were washed off and relative antigen-specific antibody levels were determined on an iQue analyzer (Intellicyt).
4
THP-1 Phagocytosis Assay for Antibody-Antigen Complexes
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THP-1 cellular phagocytosis assay was performed as previously described [15 (link)]. Briefly, biotinylated antigens were bound to FluoSphere NeutrAvidin beads (Thermo Fisher Scientific; Waltham, MA, USA). To form immune-complexes, antigen-coated beads were incubated with 10 µL of 1:100 diluted serum samples. THP-1 monocytes were added to the bead mixture and incubated at 37 °C. After 16 h incubation, cells were fixed with 4% paraformaldehyde and analyzed on an iQue analyzer (Intellicyt; Albuquerque, NM, USA).
5
Quantifying Neutrophil Phagocytosis Capacity
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Phagocytosis score of primary human neutrophils was determined as described before.30 (link) Antigens were biotinylated with NHS-Sulfo-LC-LC kit according to the manufacturer’s instruction (Thermo Fisher). Excessive biotin was removed by size exclusion chromatography using Zeba-Spin desalting columns (7 kDa cutoff, Thermo Fisher). Biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated with 1:10 diluted plasma. Primary cells were derived from Ammonium-Chloride-Potassium (ACK) buffer lysed whole blood from healthy donors and incubated with immune complexes for one hour at 37°C. For the Fc-receptor blocking experiments, isolated neutrophils were pre-incubated with 5 μg/ml of FcγR2a (CD32A, clone IV.3, Bio X Cell Cat# BE0224, RRID:AB_2687707 ) and FcγR3 (CD16, clone: LNK16, Bio-Rad, RRID:AB_324304 ) five minutes prior to addition of neutrophils to the immune complexes. Neutrophils were stained for surface CD66b (BioLegend Cat# 305112, RRID:AB_2563294 ) expression, fixed with 4% para-formaldehyde, and analyzed an iQue analyzer (IntelliCyt) (Figure S8 ).
6
Multiplex Luminex Assay for Antibody Profiling
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Antigen-specific antibody isotype and subclass titers and Fc receptor binding profiles were analyzed with a custom multiplex Luminex assay as described previously (45 (link)). In brief, antigens were coupled directly to Luminex microspheres (Luminex Corp, TX, USA). Coupled beads were incubated with diluted plasma samples washed, and Ig isotypes or subclasses with a 1:100 diluted PE-conjugated secondary antibody for IgG1 (clone: HP6001), IgG2 (clone: 31-7-4), IgG3 (clone: HP6050), IgG4 (clone: HP6025), IgM (clone: SA-DA4), IgA1 (clone: B3506B4) or IgA2 (clone: A9604D2) (all Southern Biotech, AL, USA), respectively. For the FcγR binding, a respective PE–streptavidin (Agilent Technologies) coupled recombinant and biotinylated human FcγR protein was used as a secondary probe. Excessive secondary reagent was washed away after 1h incubation, and the relative antigen-specific antibody levels were determined on an iQue analyzer (Intellicyt).
7
Phagocytosis of Antigen-Immune Complexes by THP-1 Monocytes
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THP-1 phagocytosis assay was performed as described before(48 (link)). In brief, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (Thermo Fisher) and incubated with 10 μl 1:100 diluted plasma for 2h at 37°C to form immune complexes. THP-1 monocytes were added to the beads, incubated for 16 h at 37°C, fixed with 4% para-formaldehyde and analyzed on a iQue analyzer (Intellicyt).
8
Antigen-Specific Antibody Profiling by Luminex
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Antigen specific antibody subclass, isotypes, and FcγR binding was analyzed in technical replicates by Luminex technology. Antigens were coupled to Luminex beads (Luminex Corp, TX, USA) by carbodiimide-NHS ester-chemistry with an individual region per antigen. Coupled beads were incubated with diluted plasma sample (1:100 for IgG3, IgM and IgA1, 1:500 for IgG1, FcαR and 1:2,000 for FcγR probing) for two hours at room temperature in 384 well plates (Greiner Bio-One, Germany). Unbound antibodies were washed away and subclasses, isotypes were detected with a respective PE-conjugated antibody (anti-human IgG1 (Cat# 9052-09, RRID:AB_2796621 ) , IgG3 (Cat# 9210-09, RRID:AB_2796701 ), IgM (Cat# 9020-09, RRID:AB_2796577 ) or IgA1 (Cat# 9130-09, RRID:AB_2796656 ) all SouthernBiotech, AL, USA) at a 1:100 dilution. For the analysis of FcγR binding PE-Streptavidin (Agilent Technologies, CA, USA) was coupled to recombinant and biotinylated human FcγR2a, FcγR2b, FcγR3a, FcγR3b or FcαR protein (Duke Human Vaccine Institute Protein Production Facility). Coupled FcR were used as a secondary probe at a 1:1000 dilution. After one hour incubation, excessive secondary reagent was washed away and the relative antibody concentration per antigen determined on an iQue analyzer (IntelliCyt). Each sample was analyzed in duplicates.
9
Complement Deposition Assay Protocol
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Complement deposition was performed as described before(46 (link)). In brief, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (Thermo Fisher) and to form immune-complexes incubated with 10 μl 1:10 diluted plasma samples for 2h at 37°C. After non-specific antibodies were washed away, immune-complexes were incubated with guinea pig complement in GVB++ buffer (Boston BioProducts, MA, USA) for 20 min at 37°C. EDTA containing PBS (15mM) was used to stop the complement reaction and deposited C3 on beads was stained with anti-guinea pig C3-FITC antibody (MP Biomedicals, CA, USA, 1:100, polyclonal) and analyzed on an iQue analyzer (Intellicyt).
10
Neutrophil Activation via ADNP Immune Complexes
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For the ADNP assays40 (link), biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (ThermoFisher). Immune complexes were formed by incubating antigen coupled beads with 10 ul of 1:50 diluted plasma samples at 37 °C for 2 hours. Primary neutrophils were isolated as previously mentioned and 50,000 cells per well incubated with washed immune complexes at 37 °C for 1 hour. Following incubation, neutrophils were stained for surface CD66b (Biolegend, 1:100, clone: G10F5) expression and fixed with 4% para-formaldehyde. Analysis was done on an iQue analyzer (Intellicyt).
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