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.

将A549、HepG2及U937细胞（传代2~30代）置于含盐类与葡萄糖的无血清培养基中，暴露于水溶性野生型（Wild Type, WT）提取物。该培养基配方为50 mM 羟乙基哌嗪乙硫磺酸（HEPES）、100 mM 氯化钠（NaCl）、5 mM 氯化钾（KCl）、2 mM 氯化钙（CaCl₂）及5 mM 葡萄糖，使用前调节pH至7.4以维持渗透压。水溶性野生型提取物的终浓度为0.02 mg/mL；空白对照提取物（对照组）遵循相同实验流程，仅以无菌水替代提取物。所有细胞系均接受两种暴露方案之一：单次暴露（0.02 mg/mL单次给药），或重复暴露方案：每隔一天给予0.02 mg/mL野生型提取物，共四次暴露，两次暴露之间设置恢复期。每次暴露持续24小时后，使用完全培养基洗涤细胞以清除残留的野生型提取物，随后更换新鲜培养基孵育24小时，直至下一次暴露。采用膜联蛋白V（Annexin V）-PE/7-氨基放线菌素D（7-AAD）染色法（Guava Nexin试剂，Guava Technologies公司），在488 nm激发光/575 nm发射光波长下检测细胞死亡类型。