Assessment of TNF-α Production From Human Cells

Heparinized venous blood from healthy donors was collected in accordance with the protocol approved by the Institutional Review Board for Human Subjects at Montana State University. All donors provided written consent to participate in the study. For whole-blood experiments, 20 μL of Roswell Park Memorial Institute medium (RPMI) only or RPMI with 2 × 10⁶ colony-forming units (CFU) of bacteria was added to 980 μL of freshly drawn human whole blood in 1.5-mL Eppendorf tubes. Samples were placed on an end-over-end mixer (20 RPM, 37°C, 5% carbon dioxide) and, at designated time points, samples were stained with fluorescein isothiocyanate (FITC) mouse antihuman CD14, phycoerythrin mouse anti-human CD3, and allophycocyanin mouse antihuman TNF or immunoglobulin G1 (IgG1) isotype control (BD Biosciences). Intracellular staining was performed using a BD Pharmigen intracellular stain kit following the manufacturer’s protocol, and all samples were analyzed using a FACSCalibur Flow Cytometer (BD Bioscience). Alternatively, serum was collected by centrifugation (524g for 10 minutes), filtered using a 0.2-μm filter (PALL Life Science), and stored at –80°C until quantitation of TNF-α was performed using a TNF-α–specific enzyme-linked immunosorbent assay (ELISA, BD Biosciences).

Human neutrophils (polymorphonuclear leukocytes [PMNs]) and peripheral blood mononuclear cells (PBMCs) were isolated under endotoxin-free conditions (<25 pg/mL) as previously described [6, 12]. Purity (<1% PBMC contamination) and viability (<2% propidium iodide positive) of neutrophil preparations were assessed by flow cytometry (FACSCalibur). For co-culture assays, 7 × 10⁵ neutrophils were combined with 3 × 10⁵ PBMCs at a physiological ratio and exposed to 2 × 10⁶ CFUs of S. aureus opsonized with 50% normal human serum (NHS) in a 96-well NHS-coated plate. After bacteria were added, phagocytosis was synchronized by centrifugation (524g for 7 minutes at 10°C) and then incubated at 37°C [12]. At designated time points, samples were stained as above and analyzed using flow cytometry.

For PBMC CM experiments, 5 × 10⁶ PBMCs were challenged with 1 × 10⁷ opsonized bacteria in a 24-well serum-coated plate that was then synchronized and incubated at 37°C [12]. At designated time points, samples were filtered using a 0.2-μm filter (PALL Life Science), plated to ensure they did not contain live bacteria, and stored at 4°C for further use as CM or at –80°C for assessment of intracellular TNF-α production using ELISA as described above.