Low endotoxin bsa

6 to 8-wk-old female BALB/c mice were obtained from Charles River Laboratories. Il1rl1-deficient mice were provided by Dr. Andrew McKenzie (Medical Research Council Laboratory of Molecular Biology, UK). Il-33-deficient mice were provided by Dr. Dirk Smith (Amgen Corp.). All mice were certified to be specific pathogen–free and cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee at Benaroya Research Institute (Seattle, WA). Intradermal injections were performed as previously described^{14 (link), 15 (link)}. Briefly, 5 µg TSLP (Amgen Corp.) or 2.5 µg IL-33 (R&D Systems and BioLegend) with 2.5 µg OVA (A7642; Sigma-Aldrich) or 2.5 µg HDM (Greer Laboratories) were injected intradermally in a 100 µl volume of sterile PBS every three days for a total of 4 times. Intranasal challenges with 25 µg OVA (A7642; Sigma-Aldrich) or low endotoxin BSA (Sigma-Aldrich) or 10 µg HDM in a total volume of 30 ml PBS were performed as described previously^{32 (link), 33} . For blockade with ST2-specific mAb, mice were injected with 500 μg of control mouse IgG1 or muST2-specific muIgG1 mAb (Amgen Corp.) intraperitoneally on days 15 and 19. For i.n. blockade, 50 μg of antibody was used instead.

Male C57BL/6 mice were given i.p. injections of 100 µl/50 µg CpG B or 100 µl vehicle 24 hours prior to euthanization by extraction of hearts, followed by enzymatic release of cardiac cells (as described above) and subsequent cell-separation. Cell-separation was performed using MACS Technology (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in accordance to specifications supplied by the manufacturer. Briefly, pellets of the non-CM fraction were re-suspended in buffer containing phosphate-buffered saline (PBS; Sigma-Aldrich), 0.2 mg/ml DNAse I (Roche Applied Science, Penzberg, Germany), 0.5% low-endotoxin BSA (Sigma-Aldrich) and 1 mM EDTA (Sigma-Aldrich), filtered through pre-separation filters (30 µm, MACS, Miltenyi), before incubation with CD45 MicroBeads (MACS, Miltenyi) at 4°C for 15 minutes. After incubation, labeled cells were isolated by magnetic retraction through an MS column (MACS, Miltenyi), washed and subsequently eluted. Similar procedure was applied on the resultant cell-fraction (non-CM/non-CD45⁺) using CD31 MicroBeads (MACS, Miltenyi). Final fractions (CD45⁺, CD31⁺ and CF-enriched fraction [non-CM/non-CD45⁺/non-CD31⁺]) were collected by centrifugation, and all cell-pellets were snap-frozen in liquid nitrogen and stored at −80°C before further analysis.