Macs human monocyte isolation kit 2

PBMCs were isolated from 10 ml heparin-treated blood of heroin abuser and healthy donors by density gradient centrifugation on Ficoll-Paque Plus (GE Healthcare, Buckinghamshire, UK). The cells were centrifuged and the plasma collected and frozen at −80 °C. After two washes in phosphate-buffered saline (PBS) and over a Percoll gradient, the monocyte-enriched fraction (~84.70%) was obtained. The percentage of monocytes was evaluated by flow cytometry using CD14 as a detection marker.
Monocytes were purified (~97.72%) with MACS Human Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) from buffy coats of healthy donors obtained from GuangZhou Blood Center after centrifugation according to the above methods. Monocytes (5 × 10⁶ ml⁻¹) then were resuspended in RPMI medium 1640 (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO), plated and treated as described. The 293T cell lines were grown in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum.

PBMCs were isolated from whole heparinized blood samples from SSc patients and healthy controls or from the buffy coats of healthy controls, by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA). CD14+ monocytes were purified from PBMCs using the MACS Human Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) on the autoMACs Pro Separator (Miltenyi Biotec) according to the manufacturer’s instructions. For subsequent analysis, only cell preparations with more than 95% purity (measured by FACS analysis) for CD14+ cells were used.
For selected experiments, CD14+ monocytes purified from buffy coats were cultured in RPMI 1640 + 10% FCS (fetal calf serum, <0.5 EU/mL, Sigma-Aldrich, St. Louis, MO, USA) + 2 mM Glutamine at a concentration of 2 × 10⁶ cells/mL. Cultured cells were left untreated (medium control) or treated with one of the following stimuli: 100 ng/mL ultra-pure E. coli lipopolysaccharide (LPS, strain O111:B4, Invivogen, San Diego, CA, USA), 5 µM R848 (Invivogen), 1000 U/mL IFNα-2a (Cell Sciences, Newburyport, MA, USA), and TGF-β2 (Bio-Techne, Minneapolis, MN, USA) according to the conditions and times indicated for each experiment in the results section.