Elispot human ifnγ

Manufactured by R&D Systems

Sourced in United States

The ELISpot Human IFNγ is a laboratory equipment product for the detection and quantification of human interferon-gamma (IFNγ) secreting cells. It provides a sensitive and reliable method for measuring the cellular immune response to various stimuli.

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2 protocols using elispot human ifnγ

Quantifying Peptide-Specific CD8+ T Cells

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ELISpot Human IFNγ (R&D Systems, USA) kit was used according to the manufacturer’s recommendations. Harvested CD8+ T cells were then plated and incubated at 37 °C for 24 h in the presence of irradiated peptide-pulsed PBMCs (40 Gy) that were used as stimulator cells. As a negative control, sorted CD8 T cells were incubated with irradiated non-pulsed PBMCs. Spots were revealed as mentioned in the manufacturer’s protocol and were counted using an ImmunoSpot S5 UV Analyzer (Cellular Technology Ltd., Shaker Heights, OH). IFNγ production was expressed as the number of peptide-specific spot-forming cells (SFC) per 10⁶ CD8+ T cells after subtracting the spot counts from negative control wells.

Larouche J.D., Trofimov A., Hesnard L., Ehx G., Zhao Q., Vincent K., Durette C., Gendron P., Laverdure J.P., Bonneil É., Côté C., Lemieux S., Thibault P, & Perreault C. (2020). Widespread and tissue-specific expression of endogenous retroelements in human somatic tissues. Genome Medicine, 12, 40.

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Quantification of IFN-γ-Secreting Cells and Cytolysis Assay

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For quantitative determination of the frequency of cells releasing IFN-γ, a commercial kit (ELISPOT human IFN-γ; R&D Systems) was utilized. Briefly, 1×10⁴ T cells per well were plated in triplicate and stimulated overnight with 5×10⁴ tumor cells per well on IFN-γ pre-coated 96-well plate, then incubated with the respective capture antibodies and analyzed following the instructions of ELISpot kits. Spots were counted using an automated reader (ImmunoSpot® image analyzer). The susceptibility of ES2 and ES4 cells to cytolysis by the CAR-T-cells was evaluated using a 4-h ⁵¹Cr-release assay as described in ³⁰. Briefly, CAR-T cells were co-incubated with indicated ratios of ⁵¹Cr-labeled target cells [effector-to-target ratio (E:T)] in RPMI1640 medium in 96-well U-bottom plates at 37°C for 4 hr. Lysis was measured by ⁵¹Cr release in the medium: percent lysis = (sample release – minimum release)/(maximum release – minimum release) x100, average of duplicate samples. For all experiments, untransduced (UTD) T cells served as negative controls.

Charan M., Dravid P., Cam M., Audino A., Gross A.C., Arnold M.A., Roberts R.D., Cripe T.P., Pertsemlidis A., Houghton P.J, & Cam H. (2019). GD2-directed CAR-T cells in combination with HGF-targeted neutralizing antibody (AMG102) prevent primary tumor growth and metastasis in Ewing Sarcoma. International journal of cancer, 146(11), 3184-3195.

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