64-mm tube (PennCentury?, Philadelphia, PA).(16, 17) Particle suspension was prepared as follows: fluoresbrite microspheres, sellckchem plain yellow green with a diameter of 0.05��m, were obtained from Polyscience (Brunschwig, Basel, Switzerland). The particle dimensions were confirmed using a Zetasizer (nanoseries ZEN 3600, Malvern Instruments Ltd, Worcestershire, UK) to be of 40.4��3.1 (mean value��SEM) nm. We also characterized the charge of the NPs as ?35.2mV as it was not disclosed by the manufacturer but was anticipated as they are solubilized at a step in SDS, which is subsequently removed during manufacture. The details of NP size determination and associated discrepancies have been previously described.(18) Particles were diluted to a concentration of 6.
5��105 particles/��L in media (MEM alpha for cell lines and ALI media, as defined in Ahmad et al.,(14) for primary cells) and sonicated (1mL of the NP dispersion was sonicated with a Sonicator Q55 50-Watt, 20-kHz sonicator, 30% intensity; Qsonica, LLC, Newtown, CT) for 10sec three times. For adequate particle distribution on the sample, 25��L (1.5��107particles) of particle suspension were sprayed on each air-exposed culture (>3.6��106 particles/cm2 or 0.1��g/cm2). After particle exposure the cell cultures were incubated for further 24h, then exposed to ozone [the particle exposure was done first and the same cell cultures (snapwells) were exposed to ozone], washed with PBS, and fixed. Flow cytometry The day after particle exposure cells were harvested by trypsinization and suspended in 1.0mL Hank’s Balanced Salt Solution (HBSS) containing 2.
0��g/mL propidium iodide. The samples were then put on ice. Analysis was done using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA). Ozone exposures Exposure of airway epithelial cells to ozone at precise levels was carried out in a computer-controlled in vitro exposure chamber.(15) Briefly, the exposure system consisted of four identical exposure systems maintained in a single temperature-controlled (37��C) environmental chamber (Forma Scientific, Marietta, OH) controlled by a single desktop computer. One of these four systems was always used for an air control (0ppb ozone) while the other three could be used for exposure of cells to different ozone concentrations. Ozone was produced by bubbling medical-grade compressed oxygen through a coldspark corona discharge ozone generator (Model OZ2SS-SS, Ozotech, Yreka, CA).
The air/CO2 mixture was directed into the environmental chamber where it was warmed and humidified by bubbling through a glass water bath containing 1.5L of water thermostatically Entinostat maintained at 37��C. Upon exiting the water bath, the warm air/CO2 was mixed with the ozone/oxygen stream and then passed to a glass exposure chamber containing the cells to be exposed.