According to the manufacturer's instructions, we detected the cecal IgA concentration using RayBio® Mouse IgA ELISA Kit (RayBiotech, GA, USA). Briefly, we prepared the cecal homogenate (50 mg pooled cecal sample per 100 μl) in extraction buffer (1X PBS, 0.5% Tween-20, 0.05% sodium azide) by vigorous vortexing at 4 °C. We then centrifuged the samples (1500 g, 20 min) at 4 °C and added a 1X protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) to the supernatant. We then normalized the total protein concentration of the extracted supernatant using the Bradford microplate assay. We performed IgA ELISA with (1:50) sample dilution on the pre-coated 96-well plate and measured the OD at 450 nm. We calculated the IgA concentration of the cecal samples from the known IgA standard curve.
Raybio mouse iga elisa kit
Manufactured by RayBiotech
Sourced in United States
The RayBio® Mouse IgA ELISA Kit is an enzyme-linked immunosorbent assay (ELISA) designed for the quantitative measurement of mouse immunoglobulin A (IgA) levels in various biological samples, such as serum, plasma, and cell culture supernatants.
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2 protocols using raybio mouse iga elisa kit
1
Quantifying Cecal IgA Levels in Mice
2
Flow Cytometric Analysis of HIV-1 Env Responses
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For flow cytometric analysis, fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8 (clone 53-6.7) and phycoerythrin (PE)-conjugated anti-mouse IFN-γ (XMG1.2) were purchased from Biolegend (San Diego, CA). For Intracellular Cytokine Staining (ICS) assay, the H-2d MHC class I-restricted synthetic IGPGRAFYA(311–318) peptide, corresponding to residues 311-318 of the HIV-1 Env V3 loop, was purchased from PRIMM srl (Naples, Italy). For analysis of fecal IgA, serum IgA and IgG, the HIV-1 SF162 clade B V3 peptide (PNNNTRKSITIGPGRAFYATGD) and the V3 scrambled peptide (PNNNTRKSIFYRGAPGITATGD) were purchased from Invitrogen (Carlsbad, CA) and Genscript (Piscataway, NJ), respectively. Mouse IL-4 and IFN-γ ELISA MAXTMStandard SET kits were obtained from BioLegend. Mouse CD4+ T cell isolation kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). For the quantitative measurement of IgA antibodies in fecal samples, RayBio® Mouse IgA ELISA Kit was purchased from RayBiotech, Inc. (Norcross, GA, USA).
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