Apoptosis measurements for cells exposed to repeated low levels of biomass burning

A549, HepG2 or U937 cells (passages 2–30) were exposed to water-soluble WT extracts in serum-free medium with salts/glucose. The medium consisted of 50 mM HEPES, 100 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 5 mM glucose (pH 7.4 prior to use to maintain osmolarity). The water-soluble WT extracts concentration was 0.02 mg/mL, while blank extracts (controls) underwent the same procedures as the extracts but used water instead. The cell lines were subjected to a single exposure (one exposure of 0.02 mg/ml) or a repeated exposure of four exposures of 0.02 mg/ml WT every other day across all cell types, with recovery periods in between. After each exposure (24 hours), the cells were washed with a complete medium to remove residual WT extracts and then incubated in a fresh medium (24 hours) until the subsequent exposure.The type of cell death was assessed by Annexin V (V-PE)/7-AAD (Guava Nexin Reagent, Guava Technologies) Ex\Em at 488\575 nm.