Analysis of Cytokine Secretion

PBMC were seeded at 4.5 × 10⁵ cells per well in assay medium into 96 Well V-Bottom plates (Interlab) and incubated for 30 min. If applicable, cells were pretreated for 30 min with 1 μM SR144528 (a kind gift from Roche Pharmaceuticals, Basel, Switzerland), 5 μM WIN55212-3 (Tocris), 10 μM G protein inhibitors (NF023, NF449, gallein), or vehicle control (DMSO), and subsequently treated with HU308 or vehicle, with or without forskolin and G protein inhibitors or antagonists if applicable, in 150 μL final volume. After 12 h (multiplex screen) or 6 h (quantitative IL-6/IL-10 analysis) incubation in a humidified atmosphere at 37 °C and 5% CO₂, plates were centrifuged for 10 min at 250g, and the supernatants were collected and frozen at −80 °C. The remaining cell pellets were resuspended, and aliquots (in technical duplicate) were diluted in Trypan blue for hemocytometer counting. Interleukins 2, 4, 6, 10, 12, 13, and 17A, macrophage inflammatory protein (MIP-1α), and tumor necrosis factor (TNF-α) were detected using Cytometric Bead Array (CBA) technology (BD Biosciences) and assayed on an Accuri C6 flow cytometer (BD Biosciences) as described previously.¹³⁰ For the multiplex screen, two concentrations of standard in the lower and middle range of standard curves (78 and 625 pg/mL) were utilized as internal controls, and the mean fluorescent intensity (MFI) of each cytokine was calculated. Concentrations of IL-6 and IL-10 were interpolated using FCAP Array software (v. 3.1, BD Biosciences) from standard curves. Manufacturer stated limits of detection (LOD) were 11.2, 1.4, 1.6, 0.13, 7.9, 0.6, 0.3, 0.2, and 1.2 pg/mL for interleukins 2, 4, 6, 10, 12, 13, 17A, MIP-1α, and TNF-α, respectively.