Monocytes were isolated from whole blood, from treatment-naïve NV-AMD patients (
n = 13, 7 of which also underwent FACS immunophenotyping), and controls (
n = 8, 4 of which also underwent FACS immunophenotyping), using a negative selection method as described.
45 Briefly, PBMCs were separated from blood on a gradient (Histopaque-Ficoll; Sigma-Aldrich, Munich, Germany), washed twice at 1500 rpm for 10 minutes to remove platelets, and live cells were counted with a hemocytometer (MSUM Biochem and Biotech, Moorhead, MN) using the Trypan Blue exclusion method. Total blood monocytes, including both subsets of CD14++CD16- and CD14+CD16+ monocytes were isolated using a negative selection kit
(EasySep, Stemcell Technologies, Vancouver, Canada) according to manufacturer's instructions. Flow cytometric analysis of the isolated cells validated that the entire monocyte population of blood was represented. We normally obtained 5 × 10
5 monocytes/mL blood from both NV-AMD patients and controls, which is in accordance with the normal range. RNA was extracted from the isolated monocytes using RNA isolation reagent (TriReagent; Sigma-Aldrich) according to the manufacturer's protocol. Possible remnants of DNA were degraded (DNA-free; Ambion, Austin, TX), the RNA was purified (RNAeasy MinElute Cleanup Kit; QIAGEN, Hilden, Germany), and samples were stored at −80°C until further use. RNA concentration and quality was measured using both a spectrophotometer (NanoDrop; Thermo Scientific, Waltham, MA), and a micro-fluidics based platform and data analyzer (Bioanalyzer; Agilent, Santa Clara, CA), respectively, to ascertain purity and concentration. Samples with an RNA integrity number of 8 or higher were included.