Hpbmc

Cellular chemotaxis assays were performed using disposable 12-well Transwell polycarbonate membrane inserts with 3-µm pore diameter (Corning). One mL of CM was plated directly into the bottom wells of the plate in triplicate. Serum-free media were plated directly into the wells as a negative control. For antibody-blocking chemotaxis assays, 2 µg/mL of human MCP-1 affinity-purified polyclonal antibody (R&D Systems), 1 µg/mL of human CCL5 monoclonal antibody (R&D Systems), 3 µg/mL human IL-6 monoclonal antibody (Clone 1936; R&D Systems), and 1 µg/mL RARRES2 MaxPab rabbit polyclonal antibody against full-length human RARRES2 (Abnova) were used for neutralization. The CM was pre-incubated with antibodies for 30 min at 37°C (5% CO₂). THP-1 cells (1×10⁶; ATCC) or human peripheral blood mononuclear cells (HPBMC; Lonza), both of which are models of human monocytes, were placed onto each chamber. The monocytes were allowed to migrate into the lower chamber for 6 hours at 37°C (5% CO₂). CM was then centrifuged at 150×g for 6 minutes and the supernatants were removed. The cells were quantified by a CyQUANT cell proliferation assay kit (Invitrogen). Chemotaxis index was calculated using the following equation: Chemotaxis index = (number of monocytes in the wells of the plate − control)/total number of monocytes seeded in the top of the chamber.

Frozen human peripheral blood mononuclear cells (hPBMC) were purchased from Lonza (Basel, Switzerland) and were initially collected from healthy donors under their IRB protocol. To minimize known inter-patient variability occurring across sex, age, and ancestry,[57 (link)] hPBMCs were purchased from healthy non-smoking male donors between the age of 35–50 years old of African American ancestry. The cells were thawed and incubated for 4 hours in media (RPMI-1640 medium; Corning, Tewksbury, MA) supplemented with 10% heat-inactivated fetal bovine serum (hiFBS) and 1% penicillin-streptomycin (P/S), defined as complete media, at 37 °C, 5% CO₂. After 4 hours, CD3+ primary T-cells were isolated using Human Pan T-Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) following the manufacturer’s protocol. T-cell purity (>97%) was determined by staining the cells with anti-CD3-AF700 and quantified using a flow cytometer (FACSAria Fusion, BD Biosciences, San Diego, CA). Isolated T-cells were either used directly for experiments or frozen down in a recovery freezing medium (90% hiFBS and 10% DMSO) for later use.