Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells
Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells
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DOI：
10.1529/biophysj.106.103218
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发表时间：
2008-06-15
影响因子：
3.4
通讯作者：
Hagness, S. C.
中科院分区：
文献类型：
--
作者：
Kennedy, S. M.;Ji, Z.;Hagness, S. C.
我们进行了实验，定量研究了暴露于40 μ s脉冲电场（pef）的HL60人白血病细胞中荧光质膜完整性指示剂碘化丙啶（PI）的电穿孔摄取动力学。这些实验是通过使用校准的实时荧光显微镜和开发微比色皿实现的。微比色皿是一种专门设计的设备，用于在进行实时显微镜检查时将细胞培养物暴露在强烈的pef下。进行了有限元静电模拟，以评估微管电极之间的电场不均匀程度，使我们能够将电传导响应的趋势与电场分布联系起来。实验数据分析确定了两种不同的电穿孔摄取特征：一种特征是低水平的，在PEF暴露后立即开始减速摄取，另一种特征是高水平的，加速荧光，有时在PEF暴露后数百秒表现出来。这些荧光特征的定性性质被用来分离诱导瞬态电穿孔所需的条件，并讨论电孔的稳定性和持久性。在我们的实验条件下，确定了导致HL60s瞬态电穿孔的电场强度范围在1.6 ~ 2kv /cm之间。定量分析表明，经历瞬态电穿孔的HL60s每个细胞内化了5000 - 1.25亿个核酸结合的PI分子。最后，我们表明，电场异质性可能会引起细胞培养和单个细胞内的不对称电穿孔PI摄取。
We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 us pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.