The CTL activity was examined using an IFN-γ ELISA. Peptide-pulsed cells (1 × 104 cells/well) or pancreatic cancer cells (5 × 104 cells/well) were used as a stimulator. CTLs were co-cultured with stimulator cells in 200 μl/well of 5% AS/AIM-V on 96-well round bottom plates for 20 h. After incubation, cell-free supernatants were collected, and IFN-γ production was measured using a Human IFN-γ ELISA set (BD Biosciences) and BD OptEIA™ Reagent Set B (BD Biosciences) according to the manufacturer’s instructions.
Human ifn γ elisa set
Manufactured by BD
Sourced in United States
The Human IFN-γ ELISA Set is a laboratory equipment designed for the quantitative determination of human interferon gamma (IFN-γ) in biological samples. It provides the necessary components to perform an enzyme-linked immunosorbent assay (ELISA) for the detection and measurement of IFN-γ levels.
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Human il 2 elisa kit 2, by BD (1 mentions)
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Legendplex software, by BioLegend (1 mentions)
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Human granzyme b duoset elisa kit, by R&D Systems (1 mentions)
16 protocols using human ifn γ elisa set
1
Quantifying CTL Activity via IFN-γ ELISA
2
NK cell Stimulation Assay
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Unprimed or primed NK-92 MI cells resuspended in 500μl of cMEM-α media were seeded at 5×105 live cells/well into 24-well tissue culture plates. The cells were stimulated with 5 ng/ml of rhIL-12, rhIL-15 and rhIL-18 (Biolegend) alone or in combination for 18 h. Phorbol myristic acetate (PMA) (20 ng/ml; Sigma) plus ionomycin (1 μg/ml; Sigma) were used as positive control. Cell-free culture supernatant was collected by centrifugation at 1,000× g for 5 min and measured for IFN-γ production by Human IFN-γ ELISA Set (BD Biosciences) according to the manufacturer’s instructions. For in vitro re-stimulation, primary NK cells obtained from a clinical cohort were resuspended in R10 and then plated at 1.25×105 live cells/well into 96-well round-bottom culture plate. The cells were incubated in the absence or presence of iBP (MOI 100) for 18 h. Unstimulated or stimulated NK cells were subsequently stimulated with 5 ng/ml of rhIL-12 and rhIL-18 (Biolegend CA, USA) for 18 h. NK cells were analysed for IFN-γ production using flow cytometry.
3
IFNγ and TNF-α Detection in PBMCs
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IFNγ ELISA was performed using the human IFNγ ELISA set (BD Biosciences, cat. no. 555142) according to manufacturer’s protocol. Co-staining of human IFNγ and TNF-α in PBMCs were performed using PE-conjugated anti-IFNγ (clone 4S.B3, eBiosciences, cat. no. 12-7319-82) and APC-conjugated anti-TNF-α (BD Biosciences, cat. no. 554514) antibodies according to manufacturer’s recommendation.
4
Hypoxia Effects on CAR-T Cytokine Secretion
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SKOV-3 cells (1×105/well) were seeded in 96-well flat-bottom plate. After cultured 12 hours, CAR-T cells were added at an effector/target (E/T) ratio of 3:1, and then continuously cultured in hypoxic condition (1% O2) or normoxic condition (21% O2) for 24 hours. IL-2 and interferon γ (IFN-γ) levels in the culture supernatant were determined using the Human IL-2 ELISA Set (BD Biosciences #555190) and Human IFN-γ ELISA Set (BD Biosciences #555142), respectively.
5
Cytokine Profiling of mRNA-Transfected CAR-T Cells
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The mRNA-transfected human CAR-T cells, which were stimulated with antigenic proteins for 3, 6, or 12 hours, were pulsed with BD Golgiplug (BD Biosciences) and BD Golgistop (BD Biosciences) for the last 3–6 hours of antigenic stimulation in ALyS505N-175 and then fixed with BD Cytofix/Cytoperm (BD Biosciences) overnight at 4 °C. Cells were stained with Live/Dead Fixable Aqua viability dye, fluorescent-labeled mAbs for anti-human IFN-γ (clone 4S.B3, eBioscience), anti-human TNF-α (clone MAb11, eBioscience), anti-hIL-2 (clone MQ1-17H12, eBioscience), and anti-human CD107a (hCD107a) (clone H4A3, eBioscience). The frequency of each factor-positive cell in the gated Aqua− CD8α+ cells was analyzed by FCM, as described above. The amount of IFN-γ, TNF-α, IL-2, and granzyme-B secreted from the mRNA-transfected human CAR-T cells after 24 hours stimulation in ALyS505N-0 was determined by the Human IFN-γ ELISA Set (BD Biosciences), the Human TNF ELISA Set (BD Biosciences), the Human IL-2 ELISA Kit II (BD Biosciences), and the Human Granzyme-B ELISA development kit (Mabtech, Cincinnati, OH).
6
Cytokine Profiling in Cell Samples
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Cytokine concentrations were assessed with commercial kits. Human IFNγ ELISA Set and Human IL-2 ELISA Set (BD, USA) were measured on the Sunrise spectrophotometer and analyzed with Magellan software (Tecan, Switzerland). Interleukin (IL)-2, IL-17a, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), soluble Fas ligand (sFasL), granzyme A, and perforin were measured with the LEGENDplex Human CD8/NK Panel (Biolegend, USA) on the MACSQuant X (Miltenyi Biotec, Germany) and analyzed with Legendplex software (Biolegend, USA).
7
Cytotoxicity Assay of NK-92MI Cells
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NK-92MI cells were treated with 25KbPEI for 12 h, after which time 1 × 106 cells were co-incubated (for 4 h at 37 °C/5% CO2) with target A2780, OVCAR3, SKOV3, and MDA-MB231 cancer cells at an effector:target (E:T) ratio of 10:1. Then, the cell culture supernatants were harvested to measure secreted IFN-γ using the Human IFN-γ ELISA Set (555142, BD Biosciences, San Jose, CA). Absorbance was measured in a microplate reader (Biochrome, Berlin, Germany) at a wavelength of 450 nm.
8
CAR-T Cell Cytotoxicity Assay
9
IFN-γ Quantification in Cell Culture
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IFN-γ in cell culture supernatants was measured using Human IFN-γ ELISA Set (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol.
10
Evaluating IL-12 Modulation of Tumor-Immune Interactions
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Healthy donor PBMCs or tumor digest samples, processed as described above, were co-cultured with OVCAR3 human ovarian cancer cell line, similarly to as described in Natoli et al. 2020 . Briefly, 6000 OVCAR3 cells were seeded on the wells of a flat-bottom 96-well plate in RPMI containing L-glutamine (R8758, Sigma-Aldrich), supplemented with penicillin/streptomycin (100 ng/ml, Sigma-Aldrich) and 10% FBS (Sigma-Aldrich). After 2 hours, the medium was replaced with fully supplemented RPMI containing AdV5-hu-IL12 or empty vector control (AdV5-control) at a PFU of 1000/cell. Additional control wells were left untreated. After 2 hours of incubation, the medium was replaced to remove the virus and the tumor cells were incubated for 2 days at 37°C, 5% CO2. 300,000 healthy donor PBMCs or single cells from tumor digest samples were then added to the wells of the 96-well plate in a final volume of 200 µl per well.
Tumor cell (OVCAR3) viability was assessed by an MTT assay and flow cytometry was conducted on the suspension cells (PBMCs, TILs) after 3 days of co-culture. The supernatant was collected after 6 days of co-culture to assess IFNγ and CCL5 production using a Human IFNγ ELISA Set (BD OptEIA, 555142) and ELISA MAX Deluxe Set Human CCL5 (Biolegend, 440804), respectively, according to the manufacturers' instructions.
Tumor cell (OVCAR3) viability was assessed by an MTT assay and flow cytometry was conducted on the suspension cells (PBMCs, TILs) after 3 days of co-culture. The supernatant was collected after 6 days of co-culture to assess IFNγ and CCL5 production using a Human IFNγ ELISA Set (BD OptEIA, 555142) and ELISA MAX Deluxe Set Human CCL5 (Biolegend, 440804), respectively, according to the manufacturers' instructions.
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