Tumor cytokine multiplex immunoassay

Tumor weight was recorded and 5 µL/mg of Cell Lysis Buffer with PMSF (Cell Signaling Technology) and Halt Phosphatase Inhibitor Cocktail (Thermo Scientific) was added to the tumor. Tumors were homogenized in bead beater tubes, and the lysates were stored at −80°C until use. A multiplex immunoassay was used to determine the concentration of 32 cytokines and chemokines in the tumor lysates (MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel, Millipore) following the manufacturer’s instructions. The multiplex was read on the MAGPIX System (Millipore), and the protein concentrations were interpolated from curves constructed from the protein standards and their respective median fluorescence intensity readings (Milliplex Analyst, Millipore). Log and Z-transformation of the data was performed using SPSS V.25 (IBM) and followed by unbiased hierarchical clustering (clustering only cytokines or both animals and cytokines) using on-line tool (Next Generation Clustered Heat Maps; MD-Anderson Center, University of Texas, http://www.ngchm.net/; Euclidean distance metric and McQuitty Agglomeration; accessed January 13 2020,36).