Department of Biological Sciences, The University of Illinois at Chicago, Chicago, IL 60607, USA.
Department of Electrical and Computer Engineering, The University of Illinois at Chicago, Chicago, IL 60607, USA.
J Neurosci Methods. 2018 Jan 15;294:111-115. doi: 10.1016/j.jneumeth.2017.11.007. Epub 2017 Nov 10.
Microscope chambers that accept glass coverslips with cultured cells are often used to monitor intracellular Ca concentration ([Ca]) during cell superfusion. Unfortunately, the experimental maneuvers associated with the coverslip installation in these chambers (medium removal and re-application) trigger unintended [Ca] elevations.
To prevent these [Ca] elevations, a Petri dish insert has been constructed. The insert features a superfusion-optimized well to grow cell cultures. After this insert is removed from the Petri dish, the well retains the medium. This feature allows the inserts to be installed in microscope chambers while keeping the cells submerged at all times.
These inserts were used to test the impact of a transient medium removal from the well (an equivalent of a coverslip removal from the medium) on [Ca] in primary murine cortical neurons and astrocytes, and in HEK-293 cells. In all of these models, the medium removal/re-application caused a micromolar [Ca] spike. While in neurons this spike was caused by a Ca influx, in astrocytes and HEK-293 cells, it was caused by a Ca release from intracellular stores. After the spike, a subpopulation of neurons failed to restore low [Ca]; in 24% of the astrocytes, the spike triggered [Ca] oscillations. However, prior to the spike, [Ca] was low and uniform in all these cells.
COMPARISON WITH EXISTING METHOD(S): The new method avoids the artificially-induced [Ca] elevations that take place during the handling of glass coverslips with cultured cells.
The new method allows monitoring [Ca] without disturbing the basal [Ca] levels.
接受培养细胞盖玻片的显微镜室通常用于监测细胞灌流过程中的细胞内 Ca 浓度 ([Ca])。不幸的是，这些室中与盖玻片安装相关的实验操作（介质去除和再应用）会引发意外的 [Ca] 升高。
为了防止这些 [Ca] 升高，已经构建了一个 Petri 盘插入物。该插入物具有优化的灌流孔，用于培养细胞。从 Petri 盘取出插入物后，孔中保留了培养基。该特征允许在显微镜室中安装插入物，同时使细胞始终保持在水下。
这些插入物用于测试孔中介质的短暂去除（相当于盖玻片从介质中去除）对原代小鼠皮质神经元和星形胶质细胞以及 HEK-293 细胞中 [Ca] 的影响。在所有这些模型中，介质去除/再应用都会导致微摩尔级别的 [Ca] 尖峰。在神经元中，这种尖峰是由 Ca 内流引起的，而在星形胶质细胞和 HEK-293 细胞中，它是由细胞内储存的 Ca 释放引起的。尖峰后，一部分神经元未能恢复低 [Ca]；在 24%的星形胶质细胞中，尖峰触发了 [Ca] 振荡。然而，在尖峰之前，所有这些细胞中的 [Ca] 都很低且均匀。
新方法避免了在处理培养细胞的玻璃盖玻片时发生的人为诱导的 [Ca] 升高。
新方法可在不干扰基础 [Ca] 水平的情况下监测 [Ca]。
