Recombinant human ifnα

Human whole blood [drawn with Ethylenediaminetetraacetic acid (EDTA) as anti-coagulant] was obtained from Zen-Bio Inc. (Durham, NC). Blood samples were incubated with compounds in a sterilized U-shaped 96-well plate with a volume of 200 μL/well, and stimulated with 80 ng/mL recombinant human IFNα (BioLegend, #592706) for 16 hours in a humidified, 5% CO2 cell culture incubator at 37°C. The plasma was collected for detection of C-X-C motif chemokine 10 (CXCL10, IP10) secretion. CXCL10 was measured by Human CXCL10/IP-10 AlphaLISA Kit (PerkinElmer, #AL259 C/F) according to the manufacturer’s instructions. Inhibition data were calculated by comparison to vehicle control wells for 0% inhibition and non-stimulated control wells for 100% inhibition. Dose response curves were then generated to determine the concentration required to suppress 50% of cellular response (IC₅₀) as derived by non-linear regression analysis using GraphPad Prism.

Human whole blood (drawn with EDTA as anti-coagulant) was obtained from Zen-Bio Inc. (Durham, NC). Whole blood samples were seeded at a sterilized U-shaped 96-well plate with a volume of 200 μL/well. After one-hour incubation with serially-diluted test compounds, whole blood samples were stimulated with 80 ng/mL recombinant human IFNα (BioLegend, #592706) for 15 minutes, in a humidified, 5% CO₂ cell culture incubator at 37°C. Blood was lysed for the removal of red blood cells and fixed with Fix/Lyse buffer (BD 558049), washed, and permeabilized on ice using Perm III buffer (BD 558050). Cells were stained with anti-CD3 FITC antibody (BD 555916), AF647 anti-Stat5 (pY694) antibody (BD 612567) for 30 minutes on ice. Samples were then analyzed by flow cytometry using the Guava^® EasyCyte Instrument (EMD Millipore, Burlington, MA). Phosphorylation of cellular STAT5 was quantitated by median fluorescence intensity (MFI) after gating on the CD3-positive population.

Freshly isolated PBMCs from healthy individuals were cultured for 16 h in RPMI 1640 medium containing 20% foetal bovine serum (Sigma) in the presence of the following stimuli: 1000 U/mL recombinant human IFN-α (Biolegend) or 10 ng/mL recombinant human IL-12 (Peprotech) plus 100 ng/mL IL-15 (Peprotech). GolgiStop (Sigma) was added to the medium for 4 h for stimulation, and antihuman CD107a antibody (BD) was added for 2 h for stimulation. Cells were cultured in a medium alone as a negative control.