For serum antibody responses, mice were immunized s.c. with a total of 100 μg NP-CGG (Biosearch Technologies Inc.) in alum (Sigma-Aldrich) divided among four sites. Serum was prepared from blood collected from the retro-orbital space 14 d later. For detection of cecal IgA, cecal matter was suspended in PBS at 50 mg/ml and centrifuged at 400 g for 5 min, supernatant was collected and centrifuged again at 8,000 g for 10 min, and supernatants were used for detection. Microtiter plates were coated overnight at 4°C with 10 μg/ml NP2 or NP30-BSA (Biosearch Technologies Inc.) or 2 μg/ml anti-IgA (C10-3; BD) diluted in carbonate buffer, pH 9.6. Washing was with PBS with 0.1% Tween-20 (PBST), blocking was with PBST/5% BSA, and serum or cecal supernatants were diluted in PBST/1% BSA. Serum or cecal dilutions were incubated in the coated wells for 1 h, and bound antibodies were detected using anti-IgG1 or anti-IgA conjugated to HRP (Southern Biotech) developed with 3,3′,5,5′ tetramethyl benzidine (BioLegend). Reactions were stopped after 5 min with 2 N H2SO4. Absorbance was measured at 450 nm in a SpectraMax M3 microplate reader using SoftMax pro 7.0 (Molecular Devices). Purified mouse IgA (Southern Biotech) served as the standard for cecal samples.
Anti iga c10 3
Manufactured by BD
Anti-IgA (C10-3) is a laboratory reagent used for the detection and measurement of IgA antibodies in biological samples. It functions as an immunoassay component, providing a specific binding target for IgA. The core purpose of this product is to facilitate the analysis and quantification of IgA levels in research or diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Gr 1 rb6 8c5, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd16 cd32 2.4g2, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Transcription factor buffer set, by BD (2 mentions)
Softmax pro 7, by Molecular Devices (1 mentions)
Np cgg, by Biosearch Technologies (1 mentions)
Np30 bsa, by Biosearch Technologies (1 mentions)
Purified mouse iga, by Southern Biotech (1 mentions)
Mouse albumin elisa quantitation kit, by Fortis Life Sciences (1 mentions)
Streptavidin hrp, by BioLegend (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Mouse il 10 elisa max standard, by BioLegend (1 mentions)
C10 1, by BD (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti iga c10 3
1
Quantifying Serum Antibody and Cecal IgA
2
Comprehensive Immune Cell Profiling
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To characterize the surface phenotype, the cells were isolated and resuspended in FACS buffer. After Fc receptor blocking with anti-mouse CD16/CD32 (2.4G2, BD Biosciences) for 15 minutes at 4°C, the cells were stained with antibodies against various markers. Anti-mouse B220 (RM3-6B2), CD11c (HL3), and ROR- γt (Q31-378) were from BD Biosciences. Antibodies against CD11b (M1/70), CD45 (30-F11), CD3 (145-2C11), and Gr-1 (RB6-8C5) were from eBioscience. For IgA stain, the cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and subsequently were stained intracellularly with anti-IgA (C10-3, BD Biosciences). Transcription Factor Buffer Set (BD Bioscience) was used for ROR- γt staining. Each sample was acquired with a FACSCalibur (BD Biosciences), and the data were processed with FlowJo software (Tree Star).
3
Quantification of Intestinal Biomarkers
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ELISAs for IL-10, fecal albumin, and serum leptin were performed using the following kits according to the manufacturer’s instructions: Mouse IL-10 ELISA MAX Standard (Biolegend), Mouse Albumin ELISA quantitation kit (Bethyl laboratories), Mouse Leptin ELISA kit (ENZO life sciences).
For fecal IgA measurement, 96 well maxisorp plates (Thermo Scientific) were coated overnight at 4 °C with anti-IgA (C10-3, Pharmingen) in PBS. Plates were then washed with ELISA wash buffer (PBS containing 0.05% Tween-20 (Sigma Aldrich)) and blocked with PBS containing 10% FBS for 1 h. IgA standard (M18-254, Pharmingen) and fecal supernatants (diluted 1:10) were added and incubated for 2 h. Subsequently, plates were washed and incubated with biotin-labeled IgA primary antibody (C10-1, Pharmingen) for 1 h. Plates were washed and Streptavidin-HRP (Biolegend) was added. Lastly, TMB substrate solution (eBioscience) was added and the reaction was stopped with 1 N H2SO4. Plates were read at 450 nm.
For fecal IgA measurement, 96 well maxisorp plates (Thermo Scientific) were coated overnight at 4 °C with anti-IgA (C10-3, Pharmingen) in PBS. Plates were then washed with ELISA wash buffer (PBS containing 0.05% Tween-20 (Sigma Aldrich)) and blocked with PBS containing 10% FBS for 1 h. IgA standard (M18-254, Pharmingen) and fecal supernatants (diluted 1:10) were added and incubated for 2 h. Subsequently, plates were washed and incubated with biotin-labeled IgA primary antibody (C10-1, Pharmingen) for 1 h. Plates were washed and Streptavidin-HRP (Biolegend) was added. Lastly, TMB substrate solution (eBioscience) was added and the reaction was stopped with 1 N H2SO4. Plates were read at 450 nm.
4
Comprehensive Immune Cell Profiling
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