Shandon kwik diff staining

Human eosinophils were obtained from laboratory of Dr. Bruce Bochner (Northwestern IRB), which were isolated following a published protocol (21 (link)). Briefly, 60 mL of 0.1 M EDTA-anticoagulated peripheral blood was obtained from healthy donors. Blood was diluted with 2 volumes of 1X PBS, and eosinophils were isolated by Percoll density gradient centrifugation using 40 mL of diluted blood and 10 mL of room temperature Percoll Plus (density 1.090) (GE Healthcare, 17544502), centrifuging for 20 min at 300 × g at room temperature, which concentrated and removed mononuclear cells (lymphocytes and monocytes) and basophils at the plasma-Percoll interface. In the remaining pellets, erythrocytes were removed by hypotonic lysis in ice cold water and neutrophils were removed by immunomagnetic selection using a CD16 MicroBead kit (Miltenyi Biotec, 130-045-701). Resulting eosinophil purity was confirmed by Shandon Kwik-Diff staining (ThermoFisher Scientific, 9990700). Once purified, eosinophils were primed with 30 ng/mL of IL-5 (R&D systems) overnight prior to assay.

Following euthanasia, the trachea was exposed, and a small incision was made to cannulate a 20-gauge needle. Bronchoalveolar lavage fluid (BALF) was collected from the left lung lobe after ligating the right lung lobes. Each time 0.3–0.4 mL of ice-cold Hank’s balanced salt solution (HBSS) with 100 μM EDTA was instilled and collected for a total of three times. Total leukocytes in BALF were enumerated by counting the cells using a hemocytometer. About 20,000 leukocytes were spun, and a cytospin slide was prepared (800 rpm × 3 min) and stained with Shandon Kwik Diff Staining (Thermo Fisher Scientific) to enumerate the total and differential counts of the inflammatory cells recruited into the airways.
The right side non-lavaged lung lobes were snap-frozen by immersing in liquid nitrogen and stored at − 80 °C for western blotting and PCR. The left lung was fixed with 4% paraformaldehyde for 24 h, processed, and embedded, and 5-μm-thick tissue sections were cut using a microtome. The lung sections were processed for H&E staining (supplemental data) and immunofluorescence.