Human il 2 elisa set

UniCAR T cells were cultured with or without tumor cells in the presence or absence of 50 nM TM in a 96-well plate (E:T = 5:1). Total volume was adjusted with RPMI complete media to 200 μl. After 48 h, cell culture plates were centrifuged for 5 min at 360xg and cell-free supernatant was collected. Concentration of TNF, IFN-γ, IL-2 and GM-CSF was determined by enzyme-linked immunosorbent assay (ELISA). Human TNF ELISA Set (#555212), Human IFN-Gamma ELISA Set (#555142), Human IL-2 ELISA Set (#555190), Human GM-CSF ELISA Set (#555126) as well as BD OptEIA Reagent Set B (#550534) were obtained from BD Biosciences. In addition, intracellular staining of perforin and granzyme B was performed after 48 h as described previously.^49–51 In order to monitor UniCAR T cell numbers in co-cultures, cells were labeled with Cell Proliferation Dye eFluor®670 (ThermoFisher Scientific, #65-0840-85) according to previously published protocols.^49,52 After 24 h and 96 h of incubation, 20 μl of samples was transferred to a 96-well plate and mixed with 80 μl of 1 μg/ml propidium iodide/PBS solution (ThermoFisher Scientific, #P3566). Cell numbers were calculated using a MACSQuant Analyzer 10 (Miltenyi Biotec GmbH).

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.

Approximately 2 × 10⁴ cells were mixed with OV at an MOI of 1. After 24 h, 1 × 10⁵ PBMCs were added to each well. After 48 h, the supernatant was harvested and the level of cytokine was detected using the Human IFN-γ Valukine ELISA Kit (VAL104, Novus Bio) and Human IL-2 ELISA Set (555190, BD Biosciences).