Elispots

Manufactured by BD

ELISPOTs are a type of laboratory equipment used to detect and quantify the secretion of specific proteins, such as cytokines, by individual cells. The core function of ELISPOTs is to provide a high-throughput and sensitive method for analyzing the cellular immune response.

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ELISPOT plates BD™ ELISPOT assay system Mouse IFN-γ ELISPOT Set BD ELISPOT kit reagents and protocol BD ELISPOT Set Human IFN-γ ELISPOT kit BD ELISpot kit reagents IFNγ capture antibody-coated ELISPOT plate Mouse ELISPOT kit IFN-γ ELISPOT assay kit

2 protocols using elispots

Multiplex Cytokine Detection Assay

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Paired capture and biotinylated detection antibodies for mouse IFN-γ ELISPOTs were purchased from Pharmingen (clones R4-6A2 Cat# 551216, and XMG1.2 Cat # 554410, respectively). Biotinylated goat anti-mouse IgG, IgG1 and IgG2c secondary antibodies were purchased from Jackson ImmunoResearch (Cat# 115-065-008, 115-065-205, and 115-065-208, respectively). For monitoring protein expression on arrays, monoclonal mouse anti-polyhistidine (‘HIS’; clone His-1, from Sigma Cat # H1029) and rat anti-hemagglutinin (‘HA’; clone 3F10, anti-HA high affinity, Roche Cat# 11 867 423 001) were used. Strepatavidin-alkaline phosphatase for ELISPOTs was purchased from Pharmingen (Cat# 554065). Streptavidin-conjugated SureLight® P-3 was purchased from Columbia Biosciences (Cat# D7-2212). CpG and GpC oligonucleotides were purchased from Coley Pharmaceuticals Inc. Immunostimulatory complexes (ISCOMs, AbISCO-100) were purchased from Isconova, Sweden. Magpix beads for Luminex-based cytokine assay of murine IFNγ, IL2, IL4 and IL5 were purchased from Millipore.

Davies D.H., Chun S., Hermanson G., Tucker J.A., Jain A., Nakajima R., Pablo J., Felgner P.L, & Liang X. (2014). T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association. Journal of immunology (Baltimore, Md. : 1950), 193(4), 1812-1827.

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Assessing T Cell Responses to Antigen Presentation

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The immune responses of Th cells to APCs pulsed with peptides (10 µg/mL) or proteins (10 µg/mL) were assessed using IFNγ enzyme-linked immunospot assays (ELISPOTs; BD Biosciences).⁴⁶ (link) We analyzed the frequency of peptide-specific and IFNγ producing CD4⁺ T cells per 3 × 10⁴ of bulk CD4⁺ T cells after stimulation with peptide-pulsed PBMCs (3 × 10⁴) or 1 × 10⁴ of bulk CD4⁺ T cells after stimulation with peptide-pulsed and HLA-DR-expressing L cells (5 × 10⁴/well) or RM3 (5 × 10⁴/well).³³ (link)

Sayem M.A., Tomita Y., Yuno A., Hirayama M., Irie A., Tsukamoto H., Senju S., Yuba E., Yoshikawa T., Kono K., Nakatsura T, & Nishimura Y. (2015). Identification of glypican-3-derived long peptides activating both CD8+ and CD4+ T cells; prolonged overall survival in cancer patients with Th cell response. Oncoimmunology, 5(1), e1062209.

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