Human tnf flex set

For cytokines and exhaustion markers analysis, CAR-T cells were stimulated with irradiated 8505c-TSHR cells for 24 h. The BD Cytometric Bead Array (CBA) kit were used to quantify the secretion level of IL-2, TNFα and IFN-γ of the supernatant of CAR-T cells following manufacturer’s instruction. For intracellular cytokines staining, the Golgi plug protein transport inhibitor Brefeldin A (eBioscience, San Diego, United States) was added into the cultured medium 4 h before detection. T cells were then fixed and permeabilized with Fixation and Permeabilization kit (BD Biosciences, San Jose, United States) according to manufacturer’s instruction. The following antibodies were used: FITC mouse anti-human TSHR (Santa Cruz Biotechnology, Santa Cruz, United States), Human IL-2 Flex Set (BD Biosciences), Human TNF Flex Set (BD Biosciences), Human IFN-γ Flex Set (BD Biosciences), BV421 rat anti-human IL-2 (BD Biosciences), PE-Cy7 mouse anti-human TNFα (BD Biosciences), PE-Cy7 anti-human CD279 (PD-1) (eBioscience), eFluor 450 anti-human CD223 (LAG-3) (eBioscience). Flow cytometry was performed on a CytoFLEX LX cytometer (Beckman Coulter, Brea, United States). A moflo Astrios EQ cell sorter (Beckman Coulter) was used for cell sorting. Data were analyzed with FlowJo software (FlowJo LLC, Ashland, United States).

For human samples, venous blood was collected and the serum was separated by centrifugation at 700 × g and stored at −80°C until analyzed. The cytokines in human serum were analyzed by CBA. Briefly, IL-1β, IL-6, IL-8, MIP-1β, TNF-α, and MCP-1 concentrations of CB workers and control workers were measured. Human Soluble Protein Master Buffer Kit, Human IL-1β Flex Set (Bead B4), Human IL-6 Flex Set (Bead A7), Human IL-8 Flex Set (Bead A9), Human MCP-1 Flex Set (Bead D8), Human MIP-1β Flex Set (Bead E4), and Human TNF Flex Set (Bead C4) were used according to the manufacturer’s protocol (BD Biosciences). Data were acquired on BD Canto and BD LSRFortessa flow cytometers and analyzed by FCAP Array Software (Soft Flow Inc., Pecs, Hungary).
For animal samples, the blood was collected from angular vein after deep anesthetization with chloral hydrate. The angular vein is a small vein near the eye. The angular vein was pierced by a needle and the blood was collected. Blood and lung homogenate were centrifuged at 700 × g and then the supernatants were collected and stored at −80°C until analyzed. IL-6, IL-8, IL-1β, MIP-1β, and TNF-α were evaluated with commercially available mouse ELISA kit according to manufacturers’ recommendations (IL-6, IL-1β, MIP-1β, and TNF-α kits were from R&D Systems, Minneapolis MN and IL-8 kit was from Cusabio Biotech Company, Wuhan, China).