Bio plex protm assay

Analyses were performed in February 2016, thus three months after enrolment of the final patient with NSTI and 11 months after the final control patient. Plasma levels of IL-1β, IL-6, IL-10 and TNF-α were determined using multiplex bead assays (Bio-Rad Laboratories, Hercules, CA, USA; Bio-Plex Pro^TM assay) according to the manufacturer’s instructions. Analyses were done using the Bio-Plex^® MAGPIX^TM Multiplex Reader (Bio-Rad Laboratories, Hercules, CA, USA). The samples were diluted in 1:4 and 1:10 against a standard pool with known concentration and the mean values were calculated after correction for the diluting steps. According to the manufacturer, the detection range without dilution was: IL-1β (0.2–556 pg/mL), IL-6 (5.2–18,618 pg/mL), IL-10 (1.1–11,850 pg/mL) and TNF-α (0.2–2,059 pg/mL) with an intra-assay variation of 8% and an inter-assay variation of 10%. Measurements below detection limit were set to 0. One patient had an IL-6 level above detection range, when taking the dilution factor into account, and was set to 180,000 pg/mL.

Plasma levels of Tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-1b, IL-6, IL-8, IL-17, Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1α) were measured by using the Bio-Plex Pro^TM Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and expressed as pg/ml blood plasma. IL-17 and MIP-1α were excluded from statistical analyses because values were under the detection limit of the assay.

The measurement of interleukin 6(IL-6), soluble IL-6 receptor alpha (sIL-6Ralfa), sIL-6Rbeta, IL-8, IL-10, IL-11 IL-20, IL-32, and Pentraxin-3 (PTX3) was carried out 24 h after the application of PDT. To determine the level of cytokines, supernatants of cells on which PDT had a sublethal effect were selected, which allows the assessment of the immunomodulatory effect. Previous studies have used this procedure with OSCC and HGF-1 lines [20 (link)] and other cell lines [21 (link),22 (link)]. Concentrations were assessed in supernatants using the Bio-Plex Pro^TM Assay and Bio-Plex^® 200 System (BIO-RAD Laboratories, Inc.). The multiplex assay method is commonly used to determine the concentration of cytokines in both clinical and preclinical studies [23 (link),24 (link)]. All steps of the procedure were performed following the manufacturer’s instructions. The experiments were repeated 4 times (n = 4).