Human granzyme b duoset elisa

The FBS-free supernatant of PDA cells co-cultured with T cells was assessed for released IFN-γ and granzyme B after 72 h of co-culture, using the human IFN gamma ELISA Kit (88-7316-22, Life Technologies, Cambridge, MA, USA) and human Granzyme B DuoSet ELISA (DY2906-05, R&D, Minneapolis, MN, USA). Shed MUC1 was measured using human MUC1 ELISA kit (OncoTab Inc.). All of the ELISAs were performed on the FBS-free supernatant after 72 h of co-culture.

Human Granzyme B DuoSet ELISA was purchased from R&D Systems (#DY2906-05). ELISA kits were used according to the manufacturer’s instructions.

CD26 2/3G CAR-T-cells and CD8 2/3G control cells were placed onto a 96-well culture plate (1 × 10⁵ cells/well) as the effector cells and were co-cultured overnight with CD26-Jurkat or HSB2 cells as the target cells. For positive controls of the activated effector cells, effector cells were incubated with both 10 ng/mL of phorbol-12-myristate 13-acetate (PMA) (FUJIFILM Wako, Osaka, Japan) and 500 ng/mL of ionomycin (Merck, Darmstadt, Germany) for the same period without the target cells. CD69 expression on effector cells, which were selected from the target cells by gating on marker fluorescence GFP, was then analyzed by flow cytometry using an APC anti-human CD69 antibody, clone FN50 (BioLegend). Furthermore, concentrations of interferon gamma (IFN-γ) and granzyme B in the culture supernatants secreted by the effector cells were measured by the Enzyme-Linked Immuno Sorbent Assay (ELISA) method using a Human IFN-gamma DuoSet ELISA (R&D Systems, Minneapolis, MN) and a Human Granzyme B DuoSet ELISA (R&D Systems). The concentrations of tumor necrosis factor-alpha (TNFα) and inerleukin-8 (IL-8) were also measured by flow cytometry using a BD^TM Cytometric Bead Array (CBA) Kit (BD Biosciences) according to the manufacturer’s instructions.