RT Journal Article SR Electronic T1 Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.07.10.164202 DO 10.1101/2020.07.10.164202 A1 Simon Hess A1 Christophe Pouzat A1 Lars Paeger A1 Andreas Pippow A1 Peter Kloppenburg YR 2020 UL http://biorxiv.org/content/early/2020/07/10/2020.07.10.164202.abstract AB 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 (Neher and Augustine, 1992) with perforated patch-clamp recordings (Horn and Marty, 1988). 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.Competing Interest StatementThe authors have declared no competing interest.