Crocetti Sara, Beyer Christian, Unternährer Silvio, Benavides Damm Tatiana, Schade-Kampmann Grit, Hebeisen Monika, Di Berardino Marco, Fröhlich Jürg, Franco-Obregón Alfredo
Institute for Biomechanics, ETH Zürich, Switzerland.
Cytometry A. 2014 Jun;85(6):525-36. doi: 10.1002/cyto.a.22461. Epub 2014 Mar 17.
When examined, the expansion of many stem cell classes has been shown to be facilitated by mechanically-regulated calcium entry from the extracellular space that also helps direct their developmental programs towards mechanosensitive tissues such as muscle, bone, and connective tissues. Cation channels of the transient receptor potential C class (TRPC) are the predominant conduit for calcium entry into proliferating myoblasts. Nonetheless, methods to non-invasively study this calcium-entry pathway are still in their infancy. Here we show that a microfluidic configuration of impedance-based flow cytometry (IFC) provides a method to detect TRP channel expression in cells at high throughput. Using this technology we discern changes in the IFC signal that correlates with the functional expression of TRPC1 channels and coincides with cell proliferation. Pharmacological agents, mechanical conditions or malignant states that alter the expression of TRPC1 channels are reflected in the IFC signal accordingly, whereas pharmacological agents that alter cation-permeation through TRPC1 channels, or ionophores that independently increase calcium entry across the membrane, have little effect. Our results suggest that IFC detects changes in whole-cell membrane organization associated with TRPC1 activation and surface expression, rather than cation permeation through the channel per se. IFC-based technologies thus have the potential to identify living stem cells in their earliest stages of expansion without staining or chemical fixation.
研究发现，许多干细胞类别的扩增是由细胞外空间机械调节的钙内流所促进的，这种钙内流还有助于将其发育程序导向对机械敏感的组织，如肌肉、骨骼和结缔组织。瞬时受体电位C类（TRPC）阳离子通道是钙进入增殖成肌细胞的主要途径。然而，非侵入性研究这种钙内流途径的方法仍处于起步阶段。在这里，我们展示了一种基于阻抗的流式细胞术（IFC）的微流体配置提供了一种高通量检测细胞中TRP通道表达的方法。使用这项技术，我们辨别出与TRPC1通道功能表达相关且与细胞增殖一致的IFC信号变化。改变TRPC1通道表达的药理试剂、机械条件或恶性状态相应地反映在IFC信号中，而改变通过TRPC1通道的阳离子渗透的药理试剂或独立增加跨膜钙内流的离子载体则几乎没有影响。我们的结果表明，IFC检测到的是与TRPC1激活和表面表达相关的全细胞膜组织变化，而不是阳离子通过通道本身的渗透。因此，基于IFC的技术有潜力在不进行染色或化学固定的情况下，在最早的扩增阶段识别活的干细胞。
