Cytokine multiplex bead array system

Human cancer cells were infected with 20 Vp/cell of Ads described in Results section, and media were collected at 48 hours after infection. Media samples were frozen and stored at −80 °C until analysis. After intratumoral injection of a total of 1 × 10⁸ Vp of Ads described in Results section, blood was collected retro-orbitally at different time points, and serum samples were frozen and stored at −80 °C until analysis. Tumors were harvested at 3 and 15 days after injection and washed with PBS. Tumors were homogenized with micropestles (VWR, Radnor, PA) at 4 °C, and supernatants of tumor lysates were isolated via centrifugation at 2,000 rpm for 5 minutes. Human GM-CSF and IL-12p70 in media, sera, and tumor lysates were assayed by using the BD cytokine multiplex bead array system (BD Biosciences, San Jose, CA), and analyzed using a BD Facs Array instrument (BD Biosciences) according to manufacturer’s instructions.^{30 (link),31 (link)} Total protein concentration of each tumor lysate was measured by using Micro BCA protein assay kit (Thermo Scientific, Rockford, IL), and human GM-CSF and human IL-12p70 levels in tumor lysates were calculated per gram of total protein concentration. Murine GM-CSF and IL-12p70 in media of 4T1 cell cultures were measured by a similar manner.