Il 2 elisa kit

All cytokine concentrations were measured using ELISA kits. The supernatant was collected and then detected by an IL-2 ELISA kit (R &D Systems), IL-12p70-ELISA kit (R&D systems) or an IFN-γ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

SW480 cells were inoculated into a 12-well plate (2 × 10⁴ cells/well) for incubation in a 37°C incubator overnight to allow cell attachment. The Jurkat cells were transduced with control or PD-1-expressing lentivirus (GeneChem, Shanghai, China). PD-1 expression in cell surface was measured by Fluorescence-activated Cell Sorting (FACS) analysis. The Jurkat cells were activated by adding 100 ng/mL of CD3 antibody (317303, BioLegend, San Diego, CA) and 100 ng/mL of CD28 antibody (302913, BioLegend, San Diego, CA) and subsequently cocultured with SW480 cells for 72 h. The supernatants were collected and centrifugated, and the secreted interleukin-2 (IL-2) in culture medium was tested utilizing an IL-2 ELISA kit (VAL110, R&D Systems, Minnesota).

A375 cells were pretreated with DMSO (control), 25 μM of curcumin, or 30 μM of apigenin for 4 h and then cultured in the presence of 10 ng/ml of IFN-γ for 24 h. Cells were collected, washed, resuspended and 2 × 10⁴ cells/well were plated into a 12-well flat bottom plate. Melanoma cells were incubated at 37 °C in a humidified CO₂ incubator for overnight to allow attachment. Jurkat cells were infected with control or PD-1-expressing lentiviruses (GeneChem, Shanghai). Cell surface PD-1 expression was confirmed by FACS analysis. Jurkat cells were activated with 100 ng/mL of CD3 antibody (317303, BioLegend) and 100 ng/mL of CD28 antibody (302913, BioLegend), before co-culture with A375 cells for 72 h. Plates were washed with PBS and the remaining tumor cells were stained with crystal violet solution. The dried plates were scanned and staining intensity was quantified. A cytotoxicity LDH assay kit (Dojindo, Japan) was used to measure the cytotoxic activity on target cells according to the manufacturer’s instructions. Cell-free supernatants were collected and secreted IL-2 in the media were measured as per the manufacturer’s instructions using an IL-2 ELISA Kit (VAL110, R&D Systems).