Analyzing Monocyte Subsets by In Situ Hybridization

Peripheral blood mononuclear cells were isolated from whole blood via Ficoll centrifugation (GE Healthcare, IL, USA) and stained for CD14 and CD16. Afterwards, samples were washed once with PBS + 1% BSA, fixed for 30 min at 4 °C with PrimeFlow RNA Fixation Buffer (Affymetrix) and permeabilized with Permeabilization Buffer (Affymetrix) for another 30 min at 4 °C. In situ hybridization for IL-6 and IL-8 was performed using Prime FlowRNA assay kit and probe sets according to the manufacturer (Affymetrix)26 (link). In short, samples were incubated with the appropriate target probes for 2 h at 40 °C, washed twice with PrimeFlow RNA Wash Buffer, resuspended in Storage Buffer and acquired on a FACS Canto II (BD Biosciences). Gating strategy is displayed in Fig. 4B. In short, 100.000 events per sample were recorded and the monocyte population was gated using CD14 as a selection marker. Afterwards monocyte subsets were evaluated according to the respective expression of CD16. Compensation was achieved by using single stains of BD CompBead beads with the respective secondary labels.