In vitro polarization of primary human blood derived macrophages

Purpose: Generate a benchmark to assess extent of macrophage polarization in disease or infection models. Method: Monocytes were isolated from blood donors who gave informed written consent (Ethics approval number: 161/17, Philipps University Marburg, Germany) by MACS CD14 positive selection. Monocytes were cultured for 7 days in the presence of 1% human off-the-clot AB serum. On day 7, macrophage morphology was assessed by microscope, and cells were left untreated (ctrl), stimulated with 100ng/ml LPS + 20 ng/ml IFNgamma (M1) or 20 ng/ml IL4 + 20 ng/ml IL-13 for 18h. Additionally, a separate set of cells was infected with Legionella Pneumophila Corby wild type and flagellin deficient at MOI1 for 18 h. Then RNA was isolated by phenol chloroform extraction, sample quality was assessed by Bioanalyzer, and samples with a RNA integrity > 8 were sequenced on an Illumina HiSeq 1500. Overall design: 4 biological donors who gave monocytes that were for each donor differentiated into control, M1, and M2, or served as separate set for Legionella Infection.