Xl cytokine array kit

Proteome profile analysis of human cytokines and chemokines was performed with the XL Cytokine Array Kit (R&D Systems™, Minneapolis, MN, USA). ImageJ software (NIH, Bethesda, MD, USA) was used for the quantitative evaluation; the pixel density was determined and calculated. Serum-free conditioned media obtained from 2 × 10⁵ MSC-H or MSC-CA were loaded on the membranes with blotted antibodies and evaluated as recommended by the manufacturer.

Twenty micrograms of total protein lysates were run on a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose member (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 3% non-fat milk in TBS Tween 0.1% (TBST) incubated with anti-nucleolin (Abcam, ab22758, 1:1000, Cambridge, UK) and anti-β-actin (Sigma-Aldrich, A3854, clone AC-15, St. Louis, MO, USA). Proteins were detected by chemiluminescence using an anti-rabbit or anti-mouse peroxidase-conjugated antibody (Cell Signalling) diluted 1:10,000, and Clarity ECL substrate (Bio-Rad). Images were acquired with a ChemiDoc XRS+ (Bio-Rad). For IL-6 analysis in tumours, tumours were harvested in PBS 1X and cut in small fragments. Fragments were incubated 2 h at room temperature under agitation. Supernatants and fragments were separated, and supernatants were frozen and the amount of IL-6 was analysed by ELISA (R&D Systems, Minneapolis, MN, USA). For IL-6 analysis in plasma, pools of plasma from healthy, tumour-bearing mice treated by saline solution or tumour-bearing mice treated by N6L were analysed by using a protein array (XL cytokine array kit, R&D Systems #ARY028).