Cytokine analysis

CD45⁺CD14⁺TAM were purified from tumor tissue-infiltrating leukocytes and CD14⁺ monocytes were purified from healthy control by using the Beckman Coulter MoFlow^TM cell sorting system (Beckman Coulter; Fullerton, CA, USA) for the following experiments. Purified TAM or monocytes (1×10⁵/ml) were cultured with RPMI 1640 medium (Invitrogen, Thermo Fisher Scientific Inc.; Grand Island, NY, USA) and treated with mouse anti-human TREM-1 agonist monoclonal antibody (4 μg/ml, MAB1278; R&D Systems; Minneapolis, MN, USA) or with mouse IgG1 protein (4 μg/ml, MAB002; R&D Systems; Minneapolis, MN, USA) in presence of LPS (1 μg/ml, Sigma-Aldrich; St. Louis, MO, USA) for 24 hrs. After the treatment of stimulation, the supernatants were collected for analysis of cytokines such as IL-1β, IL-6, IL-8, IL-10, IL-12p70 or TNF-α with a human inflammation Cytometric Bead Array (CBA) kit (#551811, BD Biosciences; Franklin Lakes, NJ, USA), respectively. Tests were performed according to the manufacturer's instructions available online. The six bead populations are resolved in a red channel of FC-500 flow cytometer. For each set of experiments, a standard curve was generated. The results were expressed as pg/ml and then analyzed for their relative expression (control versus treated samples). The lower limit for detection for each cytokine was determined as 10 pg/ml.