Enzyme linked immunosorbent assay kit

A cytokine release assay was performed by coculture of 10⁵ T cells with 10⁵ target cells per well in triplicate in 96-well flat-bottom plates in a 200-μl volume of complete medium. In addition, wells containing T cells alone were used as negative controls. The plates were incubated at 37°C. For antigen-specific assays, triplicate wells of Nunc MaxiSorp MicroWell plates (BioLegend) were coated with 5 μg/ml HER2-Fc chimeric protein (R&D Systems, Minneapolis, MN, USA) or CD19-Fc chimeric protein (SPEED BioSystems, Rockville, MD, USA) in 200 μl of phosphate-buffered saline (PBS) overnight at 4°C. After three washings with PBS, 10⁵ nontransduced (NT) or CAR-transduced T cells were then added, followed by incubation at 37°C. After about 24 hours, cell-free supernatants were assayed for the presence of interferon γ (IFN-γ) or IL-2 with an enzyme-linked immunosorbent assay kit (BioLegend).

One milliliter of 0.9% normal saline was injected into the lungs of mice through the bronchial tubes, and 800 μL bronchoalveolar lavage fluid (BALF) was recovered in a 1.5 ml centrifuge tube. The supernatant was collected after centrifugation at 10,000 × g for 5 min. RAW264.7 cells were pre-treated with Hor (50 and 75 μg/ml) for 1 h and stimulated with LPS (1 μg/ml) for 12 h, after which the medium was collected and centrifuged at 10,000 × g for 5 min to collect the supernatant. TNF-α, IL-6, and IL-1β protein levels were determined using an enzyme-linked immunosorbent assay kit (BioLegend, San Diego, CA, United States) according to the manufacturer’s instructions.

Pleural effusion samples were obtained using a standard thoracentesis procedure within 24 h of the patients being hospitalized. Serum samples (20 ml) were obtained from venous blood collected at the same time as the pleural effusion samples. Pleural effusion and serum samples were centrifuged with 500 U/ml heparin at 200 × g for 5 min. The supernatants were collected and frozen at −80°C until required. IL-33 concentration was detected using an enzyme-linked immunosorbent assay kit (Biolegend Inc., Chicago, IL, USA) according to the manufacturer’s instructions. All samples were analyzed twice.