Multiplex technology

The concentrations of 27 cytokines, chemokines, and growth factors in the serum samples and tumor lysates of patients with lung cancer were analyzed using multiplex technology (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol. Briefly, proteins were quantified using a Pierce BCA protein assay kit (Thermo Scientific). Bioplex Manager software (version 6.1, Bio-Rad Laboratories) was used for data acquisition and analysis.

Snap frozen primary tumor and LN samples were mechanically disrupted and treated by lysis solution (Bio-Rad). Protein concentration in lysates was determined using Bradford assay (Bio-Rad). After sonication, samples were centrifuged at 4,500 g for 6 min at 4 °C. Protein concentration in the supernatant was determined using Bradford assay and adjusted to 1000 μg/ml using serum diluent (both Bio-Rad). Amounts of inflammatory factors in tissue lysates (obtained from tissues from mice treated for 5 days and sacrificed 5 days later) were measured by multiplex technology (Bio-Rad).Transforming growth factor (TGF)-β1 was measured using a single plex kit (Millipore) according to manufacturer's protocol.