Fraction 5

Apoptosis was detected using the ApoDETECT Annexin V-FITC Kit (Life Technologies, Carlsbad, CA), following the manufacturers protocol. Briefly, cells were seeded in 6-well plates at 1 × 10⁶ cells/well in medium containing 5% FBS. Cells were treated with indicated agents for 18 hr, collected by centrifugation and washed with PBS and stained with Annexin V and propidium iodide (PI) using 1× Annexin binding buffer (provided in the kit). Following, the cells were washed with 1× binding buffer and fixed in 2% paraformaldehyde overnight. Cells were then washed in binding buffer and the percent of Annexin V-positive cells determined by flow cytometry. Data was assessed using FCS Express 4, from De Novo Software (Glendale, CA). Apoptosis was also determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei following published protocol [20 (link)] . Cells were seeded as above and treated with the indicated agents for 24 hr. Cells were collected by centrifugation, washed with ice-cold PBS containing 0.2% bovine serum albumin, Fraction V (Sigma Chemical Company), and DNA was stained for 2 hr in the dark with 0.5ml hypotonic PI buffer (0.1% sodium citrate, 50 μg/mL DNase-free RNase, 0.1% Triton X-100). DNA content was analyzed using a FACScan, and sub G0 (apoptosis marker) was quantitated using FCS Express 4 software.

Medium consisting of Eagle’s Minimum Essential Medium (EMEM; ATCC 30-2003) supplemented with 3% bovine serum albumin (Fraction V, Sigma Chemical Co.), 1.1 mM lactic acid, and 0.1 mM pyruvic acid was adjusted to pH 7.4. A cocktail of various defatting agents (10mM forskolin; 1mM GW7647; 10 mM hypericin; 10 mM scoparone; 0.4 ng/mL visfatin; 1 mM GW501516) was added to the medium/perfusate to prepare basal medium for ex vivo perfusions (10 (link)). To prepare vehicle controls, the defatting agents were replaced by an equivalent amount of dimethylsulfoxide (DMSO) as the solvent for the defatting cocktail. To make the final concentration of 12.5 ng/ml for GDNF, 50 μl of GDNF in PBS, from the GDNF stock of 100 μg/ml was added to 400 ml of the final perfusate. For ex vivo perfusions, perfusate was equilibrated with a humidified 95% O₂/5% CO₂ gas mixture.

For NO₂ exposure, a single 1-hour dose of 15 ppm NO₂ was administered (12 (link)) and mice were analyzed at several times thereafter. Comparisons were made between mice exposed to NO₂ or subjected to time in a similar exposure chamber through which HEPA-filtered room air was flowed. For NO₂-promoted allergic sensitization, a single 1-hour exposure to 15ppm of NO₂ on day 1 was followed by 30 minutes of nebulized 1% OVA, Fraction V (Sigma-Aldrich, St. Louis, MO) in saline, on days 1, 2, and 3 (29 (link)). All mice were OVA-challenged on days 14, 15, and 16, as described (30 (link)). Analyses were performed at 48 hours after the final OVA challenge, on day 18.