Mirnaeasy micro kit

After FACSorting and subsequent activation (see previous paragraph), cells were pelleted, washed once with PBS, and suspended in 700 uL buffer/sample, vortexed for 15 s, and immediately frozen at −80 °C until RNA isolation according to the miRNAeasy Micro Kit (Qiagen) manufacturer’s instructions, which was utilized for RNA isolation. Frozen isolated RNA was transported to Qiagen for sequencing.

Peripheral blood mononuclear cells (PBMCs) from both EDTA- and heparin-anticoagulated PB specimens were isolated using FICOLL™ density gradient centrifugation within 1.5 h after blood draw. CD14+ monocytes were obtained from PBMCs using a magnetic bead-enrichment protocol (CD14 MicroBeads, Human, Miltenyi Biotech, Bergisch Gladbach, Germany) and platform (AutoMACS, Miltenyi Biotech) following the manufacturer’s instructions. CD14+ monocytes obtained from EDTA-anticoagulated PB samples were analyzed for purity, and total RNA was extracted using the miRNA easy Micro Kit (QIAGEN, Venlo, The Netherlands) and stored for further analyses. In turn, CD14+ monocytes from heparin-anticoagulated PB samples were seeded at a concentration of 1 × 10⁶/mL in RPMI medium enriched with 1% penicillin/streptomycin (10,000 U/10 mg per ml; Biochrom, Berlin, Germany), 2 mM L-Glutamine, and 10% fetal bovine serum for 6 days, of which the last 48 h were under M1, M2, or vehicle (M0) stimulation. M1 polarization was performed using 100 U/mL of human recombinant interferon (IFN)-γ (R&D Systems, Minneapolis, MN, USA) and 100 ng/mL of lipopolysaccharide (LPS) from Escherichia coli (055:B5 serotype, Sigma, Burlington, MA, USA), whereas M2 polarization was performed using 20 ng/mL of interleukin-(IL)-4 plus IL-13 (R&D Systems).

Total RNA from cells and EV subpopulations was isolated using the miRNAeasy micro kit (Qiagen, Hilden, Germany), according to the manufacturer´s instructions. RNA samples were eluted in 14 μL of RNAse-free water, aliquoted, and stored at -80 °C. The quality and sized of the isolated RNA was measured by capillary electrophoresis using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA from each sample was denatured at 72 °C for 2 min and loaded into RNA 6000 Nano and Pico total RNA kits (Agilent Technologies) to analyze RNA profile and concentration. The RNA concentration was used as a normalizing loading factor for all EV samples.