Samples for intracellular staining were additionally fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilisation Kit (BD Biosciences) according to the manufacturer’s instructions. FACS acquisition was performed on LSR-II (Becton-Dickinson) and results were analysed using FlowJo software (TreeStar

Inc, Ashland, OR). RNA was isolated using an RNeasy Micro Kit (Qiagen, Hilden, Germany). Complementary DNA synthesis was carried out with an iScript Kit (Bio-Rad, Munich, Germany) and quantitative PCR was performed Selleck Doxorubicin using the following primers: S100A12: forward primer 5′-CAC ATT CCT GTG CAT TGA GG-3′, reverse primer 5′-TGC AAG CTC CTT TGT AAG CA-3′; S100A8: forward primer 5′-TGT CTC TTG TCA GCT GTC TTT CA-3′, reverse primer 5′-CCT GTA GAC GGC ATG GAA AT-3′; S100A9: forward primer 5′-GGA ATT CAA AGA GCT GGT GC-3′, reverse primer 5′-TCA GCA TGA TGA ACT CCT CG-3′; cyclophilin A: forward primer 5′-ATG CTC AAC CCC ACC GTG T-3′, reverse primer 5′-TCT GCT GTC TTT GGG ACC TTG TC-3′. Reactions were performed in triplicate using iQ SYBR Green Supermix (Bio-Rad) and normalized to endogenous cyclophilin A mRNA level using the ΔΔCt method. Lysates from FACS sorted CD14+ HLA-DR−/low MDSC and CD14+ HLA-DR+ monocytes were denatured

at 95° for 5 min and subjected to SDS–PAGE. The gel was blotted onto nitrocellulose Smad inhibitor membrane followed by incubation with anti-S100A12 antibody (Abcam, Cambridge, UK) or a control anti-glyceraldehyde 3-phosphate dehydrogenase antibody

(Sigma, St Louis, MO). Binding of the antibodies was visualized using horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam). Western blot imaging and quantitative analysis were performed using FluorChem HD2 Multiplex Fluorescent Imaging System (Cell Biosciences Inc., Santa Clara, CA). All the statistical analyses were based on two-tailed Student’s t-test. All P-values < 0·05 were considered to be significant. Differential gene expression analysis was performed to identify genes expressed in CD14+ HLA-DR−/low new MDSC but not in CD14+ HLA-DR+ monocytes. Using PIQOR Immunology Microarrays (Miltenyi), we found that S100A12 was 40-fold more strongly expressed in MDSC than in monocytes (GEO database accession no. GSE32001). Real time PCR was performed on FACS-sorted MDSC (CD14+ HLA-DR−/low) and monocytes (CD14+ HLA-DR+) from peripheral blood to confirm these results. Higher S100A12 expression was seen in MDSC than in monocytes (Fig. 1a). S100 is a family of proteins including 21 calcium-binding proteins.11 Among them, S100A8, S100A9 and S100A12 are closely related. We focused on these three proteins because monoclonal antibodies for FACS and Western blotting were available for them. First, we analysed the expression of S100A8 and S100A9 genes in the PBMC of healthy donors. Both S100A8 and S100A9 were about 10-fold to 15-fold more expressed in MDSC than in monocytes (Fig.