Easysep human monocytes isolation kit

Peripheral blood mononuclear cells (PBMCs) from CAD patients and control individuals were isolated by Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare Bioscience). The PBMCs were cryopreserved for <3 months at −80°C in 90% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) and 10% DMSO (Sigma-Aldrich). Monocytes were isolated using the EasySep Human Monocytes Isolation Kit (Stemcell Technology) and cultured in RPMI-1640 (Lonza) containing 2 mM glutaMAX (Gibco), 20 μg/mL gentamicin (Gibco), and 2% normal human serum type AB (Invitrogen).

PBMCs were thawed and monocytes were isolated using EasySep™ human monocytes isolation kit (STEMCELL Technologies Inc., Vancouver, BC, Canada), plated, and differentiated into macrophages for 8 days. M-CSF was used to stimulate MDM at a concentration of 50 ng/mL (STEMCELL Technologies Inc., Vancouver, BC, Canada). To induce foam cell formation, adherent macrophages were treated with 100 μg/mL oxLDL (Kalen Biomedical, Montgomery Village, MD, USA) for 24 h then double-stained with 0.4% oil red O for detection of lipids and anti-CD68 antibody for detection of macrophages. Double-stained CD68⁺/Oil Red O⁺-positive cells are considered foam cells. The percentage of macrophages that were also positive for oil red O was quantified in five 20× fields. Each point on the graph corresponds to the average value calculated from five different images captured for each sample. Antiretroviral drugs used were tenofovir (1 μg/mL), emtricitabine (2 μg/mL), and raltegravir (0.1 μg/mL) (Cayman chemical company, Ann Arbor, MI, USA). HIV tat, nef, and gp120 were used at a concentration of 100 ng/mL. Macrophage tropic HIV-ADA was used to infect MDMs at a concentration of 10 ng/mL of p24, a kind gift from Dr. Peter Gaskill (Drexel University School of Medicine, Philadelphia, PA, USA).