Ip 10

The up-regulated Alzheimer’s disease-specific cytokine profile of IFNγ, IP-10, and IL-9, as determined by the multivariate modeling, was applied to primary neuron and astrocyte cultures derived from CD1 P0 neonates. The combination cytokine treatment was applied 72 hours prior to experimentation in levels proportional to the concentrations we measured in 5xFAD 180-day hippocampus samples. Concentrations were centered in the nanomolar range previously used to study acute cytokine responses in neuron cultures.^{23 (link),89 (link)} Recombinant murine cytokines were purchased from Peprotech, IFN-γ (cat 315-05), IP-10 (cat 250-16), and IL-9 (cat 219-19), and reconstituted to 1 mg/mL in sterile water and diluted to 250 μg/mL (IL-9) or 10 μg/mL (IFNγ and IP-10) in 0.1% cell-culture grade bovine serum albumin (Sigma A9418) in 1X PBS. 5nM IFNγ, 12nM IP-10, and 500nM IL-9 or an equal amount of 0.1% bovine serum albumin vehicle were added to the appropriate neuronal or glial cell culture medium for treatment. Neuron cultures were treated 9 or 10 days after plating, and astrocytes were treated, following the removal of non-adherent cells, at 50% confluency. After a 72-hour stimulation, cells were assayed on the Seahorse XFe24 Extracellular Flux Analyzer or lysed for RNA isolation.

Human Tregs from healthy donors were isolated by cell sorting (Sony Sorter, MA900) based on CD3, CD4, CD25^hi, and CD127^lo expression. Tregs were seeded at 1 × 10⁴ cells in 96-well plates and then stimulated with CD3/CD28 Dynabeads (Thermo Fisher Scientific) alone or in the presence of recombinant IL-1β, IP-10, IL-6, IFN-γ, and IFN-λ2 (10 μg /mL; Peprotech) for 72 hours. Notch1 expression on Foxp3⁺ Tregs was then assessed by flow cytometry.

The human breast cancer cell lines MDA-MB-231 (ATCC® HTB-26™), BT549 (ATCC® HTB122™), HCC1937 (ATCC® CRL 2336™), MDA-MB-468 (ATCC ® HTB-132™), (American Type Culture Collection, Manassas, VA), SUM149, and SUM159 (Asterand Bioscience, Detroit, MI now acquired by BioreclamationIVT, Westbury, NY) were authenticated using a panel of microsatellite markers. Cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO₂ as previously described in Turdo et al. [3 (link)]. For stimulation experiments, MDA-MB-231 cells were starved in serum-free medium for 24 h and then treated for 48 h with a pool of 5 WHFs at a final concentration of 5% as described [23 (link)] or with PDGF-BB, Mib1b, MCP1, IP10, Il1ra, Il1b, G-CSF, Il8, Il6, EGF, FGF, Heregulin, PDGF-AA, PDGF-AB (PeproTech, Rocky Hill, NJ) at 50 ng/mL. Cells were treated in indicated experiments with cycloheximide (1 μM) or UO126 (2 μM), both of which were dissolved in DMSO (maximum concentration 0.1%) (Sigma-Aldrich).

Cell growth was assessed with CellTiter 96 AQ_ueous Non-Radioactive assay (Promega) accordingly to manufacturer's recommendations. Cells were seeded in 96-well plates with or without IP-10 (10-600 ng/mL; PeproTech) in triplicates for each condition. After 4 days of culture, MTS solution was added and absorbance measured after 3 hr.