Isotype matched control igg

Fluorescently labeled antibodies (Abs) to CD3 (145-2C11), CD25 (PC51), CD44 (IM7), CD16/CD32 (2.4G2), CD14 (rmC5-3), CD45R/B220 (RA3-6B2), and IgG2a isotype control were purchased from BD Biosciences (San Jose, CA). Fluorescently labeled Abs to GATA3 (TWAJ) and IgG1 isotype control were purchased from eBioscience (San Diego, CA). Fluorescently labeled Annexin-V, IgG2b isotype control, and 7-AAD viability staining solution were purchased from BioLegend (San Diego, CA). Ghost Dye™ Red 780 was purchased from TONBO Biosciences (San Diego, CA). Recombinant mouse proteins, including IL-2, IL-7, IL-25, TSLP, and IFN-β were from R&D Systems (Minneapolis, MN). Mouse IL-33 and IFN-α2 were from eBioscience, and mouse IFN-γ was from PeproTech (Rocky Hill, NJ). High molecular weight poly (I:C), CpG A (ODN1585), and R848 were from InvivoGen (San Diego, CA). The culture filtrate extract of A. alternata was from Greer Laboratories (Lenoir, NC); the extract contained detectable, but minimal, amounts of endotoxin (i.e., 3 ng endotoxin/mg extract). Anti-IFNAR1 (MAR1-5A3) and isotype-matched control IgG for blocking experiments were purchased from BioXcell (West Lebanon, NH).

Solutions with mast cell stabilizers ketotifen fumarate (Sigma-Aldrich) and cromolyn (Alfa Aesar) were freshly prepared prior to each treatment by diluting in sterile water, followed by passage through a 0.22-μM syringe filter. Mice were orally gavaged with 10 mg/kg of ketotifen or an equal volume of water daily, beginning 1 day post tumor implantation, and continuing until day 5 post tumor. For cromolyn, mice were intraperitoneally injected with 10mg/kg cromolyn or equal volume PBS once per day, beginning 3 days prior to 12 days-post-tumor initiation with BRPKp110 or PyMT tumors. For CCL2 inhibition, mice were intraperitoneally injected with 200 μg of monoclonal anti-mouse CCL2 (Bio X Cell) or 200 μg of the isotype matched control IgG (Bio X Cell) once per day at 8, 6, 4, and 2 days prior to the implantation of BRPKp110 or PyMT tumors.