Reproductive and Developmental Toxicology Branch, Public Health and Integrated Toxicology Division, Center for Public Health and Environmental Assessment, Office of Research and Development U.S. Environmental Protection Agency, Research Triangle Park, NC, USA.
Neurological and Endocrine Toxicology Branch, Public Health and Integrated Toxicology Division, Center for Public Health and Environmental Assessment, Office of Research and Development U.S. Environmental Protection Agency, Research Triangle Park, NC, USA.
Methods Mol Biol. 2020;2118:415-436. doi: 10.1007/978-1-0716-0319-2_29.
Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. A FACSCalibur™ flow cytometer and a Stratedigm S-1000 flow cytometer were used to measure changes in light scatter from cells after incubation with either silver nanoparticles (AgNP) or TiO nanoparticles. Within the range of between 0.1 μg/mL and 30 μg/mL the nanoparticles caused a proportional increase of the side scatter and decrease of the forward scatter intensity signals. At the lowest concentrations of TiO (ranging between 0.1 μg/mL and 0.3 μg/mL), the flow cytometer can detect as few as 5-10 nanoparticles per cell. The influence of nanoparticles on the cell cycle was detected by nonionic detergent lysis of nanoparticle incubated cells that were stained with DAPI or propidium iodide (PI). Viability of nanoparticle treated cells was determined by PI exclusion. Surface plasmonic resonance (SPR) was detected primarily in the far-red fluorescence detection channels after excitation with a 488 nm laser.Our results suggest that the uptake of nanoparticles within cells can be monitored using flow cytometry. This uptake of nanoparticle data was confirmed by viewing the nanoparticles in the cells using dark-field microscopy. The flow cytometry detection of nanoparticles approach may help fill a critical need to assess the relationship between nanoparticle dose and cellular toxicity. Such experiments using nanoparticles could potentially be performed quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.
由于在细胞内观察和测量纳米颗粒的能力有限，因此对人造纳米材料的潜在危害进行评估受到了阻碍。使用 FACSCalibur™流式细胞仪和 Stratedigm S-1000 流式细胞仪测量了细胞与银纳米颗粒（AgNP）或 TiO 纳米颗粒孵育后光散射的变化。在 0.1μg/mL 至 30μg/mL 的范围内，纳米颗粒引起侧向散射的比例增加和前向散射强度信号的减少。在 TiO 的最低浓度（在 0.1μg/mL 至 0.3μg/mL 之间）下，流式细胞仪可以检测到每个细胞多达 5-10 个纳米颗粒。通过用 DAPI 或碘化丙啶（PI）染色的经纳米颗粒孵育的非离子洗涤剂裂解细胞来检测纳米颗粒对细胞周期的影响。通过排除 PI 来确定纳米颗粒处理的细胞的活力。在用 488nm 激光激发后，主要在远红荧光检测通道中检测到表面等离子体共振（SPR）。我们的结果表明，可以使用流式细胞术监测细胞内纳米颗粒的摄取。通过使用暗场显微镜观察细胞中的纳米颗粒，证实了纳米颗粒摄取的数据。纳米颗粒的流式细胞术检测方法可能有助于填补评估纳米颗粒剂量与细胞毒性之间关系的关键需求。使用纳米颗粒进行此类实验可以使用流式细胞仪快速轻松地进行，以测量纳米颗粒摄取和细胞健康。
