Figure 2.

Diffusion of doxorubicin (DOX) through culture inserts with 3 μM pores. Neutrophils (5 × 10⁶) stimulated with 2 μM PMA or 10 μg/ml LPS were suspended in 2 mL of HBSS without phenol red and placed in the bottom chambers and cultured for 4 h to form NETs. Freshly isolated neutrophils (5 × 10⁶/2 mL) were placed at 4 °C and used as unstimulated neutrophils. DOX (10 μM) was added and incubated for another 30 min. Then, culture inserts with pores containing 2 mL of HBSS were placed in the bottom chambers. After 1 (A) and 3 (B) hours, 100 μl of medium was collected from the upper chambers and fluorescence intensity measured. The relative ratios of the fluorescein intensities were calculated compared to the control well which did not contain neutrophils. Data are shown as mean ± standard deviation in 3 different experiments. ∗: p < 0.05, ∗∗: p < 0.01.