Mouse cytokine array panel a ary006

Serum samples were obtained from blood samples of 8 weeks old mice, allowed to clot for 30 min at room temperature, and then centrifuged at 3000 × g for 15 min. Serum aliquots (150 μL) were immediately assayed for cytokines and chemokines expression by using Mouse Cytokine Array Panel A (ARY006) (R&D System, Minneapolis, USA), according the manufacturer’s conditions. Cytokines/chemokines expression levels were calculated as arbitrary units by acquiring images of developed X-ray film of cytokine array with an Image Scanner (EPSON), and measuring pixel densities of the spots with ImageJ software (ImageJ, USA).

Recombinant murine CXCL10 (250-16), CXCL13 (250-24), G-CSF (250-05), and GM-CSF (315-03) were purchased from PeproTech (Rocky Hill, NJ, USA). Directly conjugated anti-mouse mAbs CD11b-FITC (M1/70), CD45-PE/Cy7 (30-F11), Ly6G-APC/Cy7 (1A8), Ly6C-PE (AL-21) were from BD Pharmingen (San Jose, CA, USA). Carboxyfluorescein succinimidyl ester (CFSE) was from Molecular Probes (Eugene, OR, USA). The Annexin-V-APC apoptosis analysis kit (AO2001-11P-G) was obtained from Sungene (Tianjin, China). The mouse cytokine array panel A (ARY006) was purchased from R&D Systems (Minneapolis, MN, USA). The MDSC isolation kit was from Miltenyi Biotec (Bergisch Gladbach, Germany) and the streptavidin particles were from BD IMag (San Jose, CA, USA). Anti-p-Akt (Ser473, #4060), anti-Akt (#4691), anti-p-mTOR (Ser2448, #5536), anti-mTOR (#2983), anti-p-STAT3 (Tyr705, #9145) Abs were purchased from Cell Signal Technology (Danvers, MA). Anti-STAT3 (F-2) Abs was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rapamycin (HY-10219) and Stattic (HY-13818) were purchased from Med Chem Express (New Jersey, USA).