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Mouse ifn gamma elispot kit

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The Mouse IFN-gamma ELISpot Kit is a laboratory assay used to detect and quantify mouse interferon-gamma (IFN-gamma) secreting cells. It provides a standardized and sensitive method for measuring the frequency of antigen-specific T cells and monitoring immune responses.

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Lab products found in correlation

Ifnγ elispot kit plates, by R&D Systems (2 mentions) Tumor dissociation kit, by Miltenyi Biotec (2 mentions) Ifnγ elispot kit, by R&D Systems (2 mentions) Elispot reader, by Zeiss (1 mentions) Bcip nbt chromogen, by R&D Systems (1 mentions) Mouse ifnγ elispot assay, by R&D Systems (1 mentions) Inh phap, by InvivoGen (1 mentions)

7 protocols using mouse ifn gamma elispot kit

1

Tumor-Infiltrating Immune Cell Assay

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Mice bearing 10-day established B1610%TRP1KO tumors were treated by Th1, Th9, or Th17 cell ACT as described above. Mice were sacrificed on day 20 after ACT and tumor tissues were minced and digested by tumor dissociation kit (Miltenyi Biotec). Each host immune cell subset in about 200 mg tumor tissues was isolated by a bead positive selection kit (CD8, NK1.1, or CD4). Isolated cells per 200 mg tumor tissues were cocultured with irradiated B16TRP-1KO tumor cells on IFNγ ELISpot Kit plates (Mouse IFN-gamma ELISpot Kit, R&D Systems) for 48 hours following the manufacturer's instructions. The plates were imaged and evaluated by Cellular Technology Limited ELISPOT Analyzer.
Xue G., Zheng N., Fang J., Jin G., Li X., Dotti G., Yi Q, & Lu Y. (2021). Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to eliminate antigen-loss-variant tumor cells. Cancer cell, 39(12), 1610-1622.e9.
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2

SARS-CoV-2 S Protein T-cell Immune Response Assay

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Analysis of the T-cell immune response was performed by using the Mouse IFN-gamma ELISpot Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Splenocytes were seeded (5 × 105 cells/well, RPMI medium with 10% FBS) and stimulated with a pool of peptides (20 μg/mL each) from the RBD of SARS-CoV-2 S protein sequence, restricted by major histocompatibility complex (MHC) class I (H2-Dd, H-2-Kd, and H-2-Ld) and MHC class II (H2-IAd and H2-IEd) molecules of BALB/c mice. The peptides were selected using the IEDB Analysis Resource instruments and synthesized by AtaGenix Laboratories (Wuhan, China). For the negative control, splenocytes were incubated without stimulation, and Concanavalin A was added to the well for the positive control. The cells were incubated for 20 h at 37 °C in the presence of 5% CO2. Subsequently, the plates were washed and incubated with the primary antibody against IFN-γ. The plates were washed again and incubated with a secondary antibody conjugated to alkaline phosphatase. Finally, the plates were washed again and incubated with BCIP/NBT. The number of IFN-γ-producing cells was counted using an ELISpot reader (Carl Zeiss, Oberkochen, Germany). The number of spot-forming units (SFU) per million cells was calculated by subtracting the average value from the negative control wells.
Volosnikova E.A., Merkuleva I.A., Esina T.I., Shcherbakov D.N., Borgoyakova M.B., Isaeva A.A., Nesmeyanova V.S., Volkova N.V., Belenkaya S.V., Zaykovskaya A.V., Pyankov O.V., Starostina E.V., Zadorozhny A.M., Zaitsev B.N., Karpenko L.I., Ilyichev A.A, & Danilenko E.D. (2023). SARS-CoV-2 RBD Conjugated to Polyglucin, Spermidine, and dsRNA Elicits a Strong Immune Response in Mice. Vaccines, 11(4), 808.
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3

Mouse IFNγ ELISPOT Assay Protocol

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Mouse IFNγ ELISPOT assay was performed according to the manufacturer instructions (R&D Systems) with minor modifications. Briefly, 005 GSCs (1 × 105) in 10% RPMI were irradiated (35 Gy) after overnight culture. Splenocytes (1–2 × 106/well) harvested from treated mice (as in (Cheema et al., 2013 (link))) were co-cultured with 1 × 105 irradiated (35Gy) 005 GSCs or FCS-differentiated 005 cells in 12 well plate for 48 hr at 37°C. Stimulated splenocytes (1–2 × 10 5) were plated onto 96-well PVDF-backed microplates coated with monoclonal antibody specific for mouse IFNγ (Mouse IFN-gamma ELISpot kit; R&D Systems). After 24 hr of incubation at 37°C, plates were washed three times, incubated with detection antibody concentrate overnight at 4°C, washed and incubated with streptavidin-AP for 2 hr at room temperature. After washing, the signal was developed with BCIP/NBT Chromogen (R&D Systems) for one hour at room temperature. Spots were identified and counted on an AID Version 3.1.1 ELISPOT reader.
Saha D., Martuza R.L, & Rabkin S.D. (2017). MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE. Cancer cell, 32(2), 253-267.e5.
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4

Evaluating CD8+ T Cell Response

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BALB/c mice were inoculated at the left mammary gland with 5×105 EMT6 tumor cells and i.v. injection with 1×105 EMT6 tumor cells (which allows lung tumor metastasis). IgG, IgG-Dye+NIR, αCD73-Dye, or αCD73-Dye+NIR treatments were given on day 5 to orthotopic tumors when the tumor size reached around 130 mm3 (other parts of mice were shielded from light). The lung metastatic tumor tissues were minced and digested. CD8+ T cells in the tissues were isolated by a bead positive selection kit (CD8). Isolated cells per 100 mg tumor tissues were cocultured with irradiated EMT6 tumor cells on IFNγ ELISpot Kit plates (Mouse IFN-gamma ELISpot Kit, R&D Systems) for 48 hours following the manufacturer’s instructions. The plates were imaged and evaluated by Cellular Technology Limited ELISPOT Analyzer.
Xue G., Wang Z., Zheng N., Fang J., Mao C., Li X., Jin G., Ming X, & Lu Y. (2021). Elimination of acquired resistance to PD-1 blockade via the concurrent depletion of tumour cells and immunosuppressive cells. Nature biomedical engineering, 5(11), 1306-1319.
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5

Evaluating TRP-1 T Cell Cytotoxicity

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B1620%TRP−1-KO cells (1×106) were s.c. injected into both flanks of B6 mice. TRP-1 T cells (5×106) or TRP-1 T10%MYXV cells (5×106) were i.v. injected on day 7 when tumors reached ~7×6 mm. In some mice, free MYXV was i.t. injected only into the tumors on the left flanks. Mice were sacrificed on ~20 days after tumor inoculation and tumor tissues were minced and digested using a tumor dissociation kit (Miltenyi Biotec). Each host immune cell subset in about 200 mg tumor tissues was isolated by a bead positive selection kit (CD8+ or CD4+). Isolated cells per 200 mg of tumor tissues were cocultured with irradiated B16TRP−1-KO tumor cells on IFNγ ELISpot Kit plates (Mouse IFN-gamma ELISpot Kit, R&D Systems) for 48 hrs following the manufacturer’s instructions. The plates were imaged and evaluated by a Cellular Technology Limited ELISPOT Analyzer.
Zheng N., Fang J., Xue G., Wang Z., Li X., Zhou M., Jin G., Rahman M.M., McFadden G, & Lu Y. (2022). Induction of tumor cell autosis by myxoma virus-infected CAR-T and TCR-T cells to overcome primary and acquired resistance. Cancer cell, 40(9), 973-985.e7.
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6

In Vivo IFN-γ Elispot Assay

Cited 1 time
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IFN-γ Elispot assays were conducted using the Mouse IFN-gamma ELISpot Kit (XEL485, R&D Systems) as according to the manufacturer’s instructions.49 (link) Spleens were collected on day 8 post-treatment. Spleen cells suspended in Roswell Park Memorial Institute (RPMI)-1640 Medium (supplemented with 10% FBS, 10 ng/mL mIL2, and 1 µM 2-mercaptoethanol) were loaded to the 96-well plates (1×105 spleen cells per well). Antitumor cell immunity was analyzed by loading live tumor cells (1×104 tumor cells per well) into the spleen cell preseeded wells. Antitumor antigen immunity was analyzed by loading tumor antigen peptides (Trp2 180–188 or gp100 25–33, a final concentration of 5 µM for each peptide) to the spleen cell preseeded wells. After tumor cells, peptides, or IL12s were loaded, the plates were cultured at 5% CO2 and 37°C for 3 days before spots were developed and counted. Phytohemagglutinin P (inh-phap, Invivogen) (100 µg/mL) was added to the positive control wells.
Jiang H., Nace R., Carrasco T.F., Zhang L., Whye Peng K, & Russell S.J. (2024). Oncolytic varicella-zoster virus engineered with ORF8 deletion and armed with drug-controllable interleukin-12. Journal for Immunotherapy of Cancer, 12(3), e008307.
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7

Evaluating CD8+ T Cell Response

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BALB/c mice were inoculated at the left mammary gland with 5×105 EMT6 tumor cells and i.v. injection with 1×105 EMT6 tumor cells (which allows lung tumor metastasis). IgG, IgG-Dye+NIR, αCD73-Dye, or αCD73-Dye+NIR treatments were given on day 5 to orthotopic tumors when the tumor size reached around 130 mm3 (other parts of mice were shielded from light). The lung metastatic tumor tissues were minced and digested. CD8+ T cells in the tissues were isolated by a bead positive selection kit (CD8). Isolated cells per 100 mg tumor tissues were cocultured with irradiated EMT6 tumor cells on IFNγ ELISpot Kit plates (Mouse IFN-gamma ELISpot Kit, R&D Systems) for 48 hours following the manufacturer’s instructions. The plates were imaged and evaluated by Cellular Technology Limited ELISPOT Analyzer.
Xue G., Wang Z., Zheng N., Fang J., Mao C., Li X., Jin G., Ming X, & Lu Y. (2021). Elimination of acquired resistance to PD-1 blockade via the concurrent depletion of tumour cells and immunosuppressive cells. Nature biomedical engineering, 5(11), 1306-1319.
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