Anti cd14 pb clone m5e2

Sorted CD14⁺ cells were incubated for 6 days at 1*10e6 cells per ml in Roswell Park Memorial Institute (RPMI; Gibco, Life Technologies) supplemented with 10 % FCS (heat inactivated, PAA Laboratories), 50 ng/ml premium grade recombinant granulocyte macrophages colony-stimulating factor (GM-CSF) and 20 ng/ml premium grade recombinant IL-4 (Miltenyi Biotec) to obtain differentiated monocyte-derived DCs (moDCs). Cell differentiation quality was checked by flow cytometry with the following antibodies: anti-CD11c APC (clone B-ly6, BD Biosciences) and anti-CD14 PB (clone M5E2, BD Biosciences). Proper differentiation was considered as completed when at least 80 % of the harvested cells were CD11c⁺CD14⁻. For mRNA analysis, moDCs were lysed with RLT Plus buffer (Qiagen) and kept at −20 °C until further extraction.

Peripheral blood mononuclear cells (PBMC) were freshly isolated by Ficoll (GE Healthcare Biosciences) as described previously [37 (link)]. PBMC subpopulations were sorted with anti-CD4, anti-CD8, anti-CD14, anti-CD19, and anti-CD56 MicroBeads (Miltenyi Biotec) with an autoMACS Pro Separator (Miltenyi Biotec) according to manufacturer’s instructions. The purity of sorted cells was checked by flow cytometry with the following antibodies: anti-CD4 APC-H7 (clone SK3, BD Biosciences), anti-CD4 ECD (clone SFCI12T4D11, Beckman Coulter), anti-CD8 FITC (clone RPA-T8, BD Biosciences), anti-CD11c APC (clone B-ly6, BD Biosciences), anti-CD14 PB (clone M5E2, BD Biosciences), anti-CD19 PE (clone HIB19, eBioscience), and anti-CD56 PE-Cy7 (clone NCAM16.2, BD Biosciences) on a LSRII flow cytometer (BD Biosciences). We did not pursue the proposed experiments if the purity of sorted cells was less than 90 %. Analyses were performed using FlowJo software (Treestar).