Journal: Heliyon

Article Title: Neutrophil extracellular traps (NETs) reduce the diffusion of doxorubicin which may attenuate its ability to induce apoptosis of ovarian cancer cells

doi: 10.1016/j.heliyon.2022.e09730

Figure Lengend Snippet: The effects of NETs on the diffusion of doxorubicin (DOX). Neutrophils (1 × 10 7 /4 ml) stimulated with PMA (A) or LPS (B) were placed in the bottom chamber as described in Figure 2 legend and DOX added at a final concentration of 10 μM and incubated for another 30 min. In some wells, DNAse I was added at a final concentration of 1000 u/ml at 30 min before the addition of DOX. Then, culture inserts containing 2 mL of HBSS were placed in the bottom chambers and auto fluorescence intensities of DOX in the upper chamber measured at the indicated time points. In each set of experiments, the relative ratios of fluorescein intensities were calculated against the value of the samples measured at 1 h after incubation in control wells which did not contain NETs and DNAse I. Data are shown as mean ± standard deviation in 3 (PMA) and 3 (LPS) different experiments. ∗: p < 0.05, ∗∗: p < 0.01.

Article Snippet: SYTOX green nucleic acid stain (≥99%) and DNAse I (≥90%) were purchased from Thermo Fisher Scientific (Waltham, MA) and Worthington Biochemical Co. (Lakewood, NJ), respectively.

Techniques: Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Standard Deviation

Journal: Heliyon

Article Title: Neutrophil extracellular traps (NETs) reduce the diffusion of doxorubicin which may attenuate its ability to induce apoptosis of ovarian cancer cells

doi: 10.1016/j.heliyon.2022.e09730

Figure Lengend Snippet: Peritoneal tumors of SKOV-3 were induced as described in Materials and Methods, and similar sized tumors (approximately 3∼5 mm in diameters) were soaked in 50 mM DOX diluted in 4 ml of HBSS buffer with unstimulated (A) or PMA-stimulated (B) neutrophils in 15 ml tube. In (C), DNase I (1000 u/ml) was added with PMA-stimulated neutrophils at the start of the experiment. After 3 h, the tumors were taken out, fixed with dry-iced acetone and 10-μM cryostat sections of post-fixed frozen samples were created. After the counterstaining the nuclei with DAPI, the infiltration of DOX from the tumor surface was evaluated with the detection of autofluorescence under fluorescence microscopy (BZ8000; Keyence, Osaka, Japan). Figures show the merged images for DOX (red) and DAPI (Blue).

Article Snippet: SYTOX green nucleic acid stain (≥99%) and DNAse I (≥90%) were purchased from Thermo Fisher Scientific (Waltham, MA) and Worthington Biochemical Co. (Lakewood, NJ), respectively.

Techniques: Fluorescence, Microscopy

Journal: Heliyon

Article Title: Neutrophil extracellular traps (NETs) reduce the diffusion of doxorubicin which may attenuate its ability to induce apoptosis of ovarian cancer cells

doi: 10.1016/j.heliyon.2022.e09730

Figure Lengend Snippet: KOC-2S or SKOV3 cells were embedded in collagen gel droplets as described in Materials and Methods and cultured in 2 ml media containing 15 μM doxorubicin (DOX) with neutrophils (1 × 10 7 ) and/or DNAse I (1000U/ml). NETs (-) and (+) show the data in the presence of unstimulated and LPS-stimulated neutrophils, respectively. After 12 h, apoptotic cells were examined with FACSCalibur. (A) Representative FACS Profiles of KOC-2S (B) Data are shown as mean ± standard deviation in triplicate from one of 4 (KOC-2S) and 3 (SCOV-3) different experiments. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001.

Article Snippet: SYTOX green nucleic acid stain (≥99%) and DNAse I (≥90%) were purchased from Thermo Fisher Scientific (Waltham, MA) and Worthington Biochemical Co. (Lakewood, NJ), respectively.

Techniques: Cell Culture, Standard Deviation