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Hess, Simon; Pouzat, Christophe; Paeger, Lars; Pippow, Andreas; Kloppenburg, Peter
Cell calcium (Edinburgh), 07/2021, Letnik: 97Journal Article
Display omitted •Combining added buffer approach and perforated patch clamp optimizes quantification of Ca2+ handling.•β-escin as perforating agent allows controlled Ca2+ buffer loading while preserving cytoplasmic pathways.•Mobile and Immobile Ca2+ buffers can be quantified. Ca2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the ‘added buffer’ approach 1 with perforated patch-clamp recordings 2. Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.
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