Lysis buffer 11

As previously described,17 (link) tumor tissue samples were homogenized and digested with Lysis Buffer #11 (R&D Systems) containing protease inhibitors on ice for 1 hour. The samples were then centrifuged at 14,000 rpm for 20 min at 4^oC, and supernatants were collected and then delivered to Eve Technologies Corporation (Calgary, AB. Canada) for cytokines/chemokines detection using multiplexed magnetic Luminex assay.

Tumor tissue samples were homogenized and digested with lysis buffer #11 (R&D Systems) containing protease inhibitors on ice for 1 hour. The samples were then centrifuged at 14,000 rpm for 20 min at 4^oC, and supernatants were collected. Blood samples were centrifuged at 3000 rpm for 5 min at 4^oC, and supernatants were collected as plasma. Cytokines and chemokines in tumor tissue or plasma were analyzed using a mouse premixed multianalyte kit according to manufacturer’s instruction (USA R&D Systems). Samples with twofold dilution were run in a 96-well plate using the customized multiplex cytokine/chemokine panels. A Bio-Rad BioPlex 200 analyzer was used to determine the mean fluorescence intensity (MFI) for 50 beads per analyte. The MFIs were compared with the standard curve using a five-parameter logistic regression analysis program.