CAR-T cells were co-incubated for 24 h with BAFF-R expressing target cells (tumor lines) at an effector-to-target (E:T) ratio of 2:1. Controls included BAFF-R-negative L cells, BAFF-R-positive B2D cells, CD3/CD28 beads (10 μL/106 CAR-T cells), and non-transduced T cells from the same donor. Supernatant was collected for ELISA detection of cytokines (Human IFN-γ ELISA Set and Human TNF ELISA Set, BD Biosciences) and granule release (Human Granzyme B DuoSet ELISA Kit, R&D).
Human granzyme b duoset elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human Granzyme B DuoSet ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human granzyme B levels in cell culture supernates, serum, and plasma.
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6 protocols using human granzyme b duoset elisa kit
1
CAR-T Cell Cytotoxicity Assay
2
TCR-transduced T cell co-culture assay
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Co-culture experiments of TCR-transduced T cells with HEK293T or U2OS were performed in 250 μL medium (RP-MI-1640 supplemented with 10% FBS, 10,000 U/mL penicillin, and 10 mg/mL streptomycin) in 48-well plates for 18 h; co-cultures with PBMC were performed in 150 μL medium in 96-well plates for 24 h at 37°C and 5% CO2. Indicated peptides (Genscript) were added at 5 μM final concentration 1 h before co-culture. For HLA-blocking, Ultra-LEAF Purified Anti-Human HLA-class I (clone W6/32, BioLegend, RRID: AB_2561492) or Ultra-LEAF Purified Mouse IgG2a K isotype control (clone MOPC-173, BioLegend, RRID: AB_11148947) were added at 50 μg/mL final concentration 1 h before co-culture. Activation of T cells was detected using the Human IFN-y ELISA Set BD OptEIA (BD Biosciences, RRID: AB_2869029), the human granzyme B DuoSet ELISA kit (R&D Systems), and by flow cytometry.
3
Evaluating IgE TRAP Fc Function
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To evaluate whether IgD/IgG4 hybrid Fc domain of IgETRAP lacks IgG Fc-mediated function unlike omalizumab, ELISA plates were coated with Nitrophenyl-hapten (NP)-BSA (Bioresearch technologies, UK; 10 μg mL−1) at 4 °C overnight and, after washing with PBS, incubated with anti-NP chimeric human IgE (JW8/1; GeneTax, USA; #GTX17414; 1:3 dilution) for 1 h at 37 °C. After washing with IMDM media containing 20% FBS, the plates were incubated with various concentrations of each drug (IgETRAP, IgETRAP-IgG1M2 or omalizumab) for 1 h at 37 °C. IgETRAP-IgG1M2, which is IgETRAP with superior ability to induce IgG1 Fc-mediated side effects, was used as a positive control. The plates were cultured with NK103 cells (1 × 105 cells) expressing FcγRIIIA for 5 h at 37 °C in CO2 incubator following a washing step with IMDM media containing 20% FBS. During the culture, NK cells were activated through FcγRIIIA to secrete granzyme B, which was used as an indicator of IgG-mediated function. Granzyme B in culture supernatant was measured with human granzyme B DuoSet ELISA kit (R&D systems, USA) according to the manufacturer’s instruction. NK103 cells are FcγRIIIA-expressing NK cell line derived from human lymphoma, NK10146 (link). NK103 was kindly provided by Dr. Sae Won Kim (SL-GIBEN Inc., Korea).
4
Quantifying Cytokine Secretion in Supernatant
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To determine the interferon-gamma (IFN-γ) and granzyme B concentration in the collected supernatant, a human IFN-γ ELISA kit from Thermo Fisher Scientific and a human granzyme B DuoSet ELISA kit from R&D Systems were used [35 (link)]. Next, 50 µL of supernatant (consisting of cell-secreted proteins) was transferred in duplo to microtiter plates that were pre-coated overnight with IFN-γ or granzyme B capture Abs. Analyses were performed according to the manufacturers’ protocol. OD-values of each well were measured with an IMARK® (Bio-rad, Belgium, Temse) microplate absorbance plate reader (λ = 450 nm). In order to determine the IFN-γ and granzyme B concentrations in our supernatant, standards (with known IFN-γ or granzyme B concentrations) were included.
5
NK Cell Activation Assay in Hypoxia
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NK cells were plated 80,000 cells per 200 µL in a 96-well plate with 200 U/mL IL-2 and pre-incubated in 21% or 1% O2 for 24 h. Corning 96-Well TC-Treated Microplates were coated with 10 µg/mL of each antibody/ligand (Recombinant Human ULBP-1, R &D Systems #1380-UL-050; UltraLeaf anti-CD16 ab (3G8), Biolegend #302057; UltraLeaf anti-NKp46 ab (9E2), Biolegend #331947) for 3 h and then washed three times with 1 × PBS. After antibody/ligand coating of the plates, the NK cells were then transferred to the coated plate and incubated for 18 h in 21% or 1% O2. After incubation, cells were spun down at 300 × g for 5 min. Supernatant was removed and stored at -20 °C until ELISA was performed. Human IFN-γ was assayed with the human IFN-gamma DuoSet ELISA kit (R&D DY285B) and granzyme B was assayed with human granzyme B DuoSet ELISA kit (R&D DY2906-05) according to manufacturer’s instructions. Concentrations were drawn from a standard curve performed on each plate.
6
Cytokine Profiling of TCR-Transduced T Cells
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A total of 60 000 TCR-recombinant 58a À b À cells were cultured with 100 000 antigen-presenting cells. Cells were co-cultured in 150 mL RPMI 1640 and 10% FBS in 96-well plates for 16 h at 37°C and 5% CO 2 . For target peptide loading, 3 £ 10 6 antigen-presenting cells were incubated with the respective target peptide at 7.5 mmol/L for 30 min prior to co-culture.
TCR-transduced human lymphocytes were cultured at 50 000 T cells with 10 000 potential target cells in 200 mL RPMI 1640 and 10%
FBS in 96-well plates at 37°C and 5% CO 2 for 20 h. Exact effector-totarget ratios of individual co-cultures depended on frequencies of CD8 + and TCR-transduced T cells within individual T-cell preparations and can be found in the supplementary material. Interferon gamma (IFN-g), granzyme B and tumor necrosis factor alpha (TNF-a) were determined in cell culture supernatants using a human IFN-g ELISA set (BD Biosciences), human granzyme B DuoSet ELISA kit (R&D Systems) and human TNF-a DuoSet ELISA kit (R&D Systems).
TCR-transduced human lymphocytes were cultured at 50 000 T cells with 10 000 potential target cells in 200 mL RPMI 1640 and 10%
FBS in 96-well plates at 37°C and 5% CO 2 for 20 h. Exact effector-totarget ratios of individual co-cultures depended on frequencies of CD8 + and TCR-transduced T cells within individual T-cell preparations and can be found in the supplementary material. Interferon gamma (IFN-g), granzyme B and tumor necrosis factor alpha (TNF-a) were determined in cell culture supernatants using a human IFN-g ELISA set (BD Biosciences), human granzyme B DuoSet ELISA kit (R&D Systems) and human TNF-a DuoSet ELISA kit (R&D Systems).
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