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Detailed practical information is provided with emphasis on mapping cytosolic and mitochondrial pH, mitochondrial Na(+), and briefly also aspects related to mitochondrial Ca(2+) measurements in living cells, as grown on (un)coated glass coverslips. This chapter lists (laser scanning confocal) microscope instrumentation and setup requirements for proper imaging conditions, cell holders, and an easy-to-use incubator stage. For the daily routine of preparing buffer and calibration solutions, extensive annotated protocols are provided. In addition, detailed measurement and image analysis protocols are given to routinely obtain optimum results with confidence, while avoiding a number of typical pitfalls.

In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+](m)) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+]m. In the first 15 min of MI, a twofold increase of [Na+]m was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+]c) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+]m dropped to 91 +/- 7 mM, whereas [Na+]c further increased to 91 +/- 4 mM. The striking rise in [Na+]m is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+]m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+]m measurements in nonpermeabilized living cells.

In ischemic or hypoxic tissues, elevated Ca2+ levels have emerged as one of the main damaging agents among other Ca2+-independent mechanisms of cellular injury. Because mitochondria, besides the endoplasmic reticulum, play a key role in the maintainance of cellular Ca2+ homeostasis, alterations in the mitochondrial Ca2+ content ([Ca2+]m) were monitored in addition to changes in cytosolic Ca2+ concentration ([Ca2+]i) during metabolic inhibition (MI) in renal epithelial Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and [Ca2+]m were monitored via, respectively, fura 2 and rhod 2 measurements. MI induced an increase in [Ca2+]i reaching 631+/-78 nM in approximately 20 min, followed by a decrease to 118+/-9 nM in the next approximately 25 min. A pronounced drop in cellular ATP levels and a rapid increase in intracellular Na+ concentrations in the first 20 min of MI excluded Ca2+ efflux in the second phase via plasma membrane ATPases or Na+/Ca2+ exchangers (NCE). Mitochondrial rhod 2 intensities increased to 434+/-46% of the control value during MI, indicating that mitochondria sequester Ca2+ during MI. The mitochondrial potential (deltapsim) was lost in 20 min of MI, excluding mitochondrial Ca2+ uptake via the deltapsim-dependent mitochondrial Ca2+ uniporter after 20 min of MI. Under Na+-free conditions, or when CGP-37157, a specific inhibitor of the mitochondrial NCE, was used, no drop in [Ca2+]i was seen during MI, whereas the MI-induced increase in mitochondrial rhod 2 fluorescence was strongly reduced. To our knowledge, this study is the first to report that in metabolically inhibited renal epithelial cells mitochondria take up Ca2+ via the NCE acting in the reverse mode.

In ischemic or hypoxic tissues, elevated Ca2+ levels have emerged as one of the main damaging agents among other Ca2+-independent mechanisms of cellular injury. Because mitochondria, besides the endoplasmic reticulum, play a key role in the maintainance of cellular Ca2+ homeostasis, alterations in the mitochondrial Ca2+ content ([Ca2+]m) were monitored in addition to changes in cytosolic Ca2+ concentration ([Ca2+]i) during metabolic inhibition (MI) in renal epithelial MadinDarby canine kidney (MDCK) cells. [Ca2+]i and [Ca2+]m were monitored via, respectively, fura 2 and rhod 2 measurements. MI induced an increase in [Ca2+]i reaching 631 ± 78 nM in ∼;20 min, followed by a decrease to 118 ± 9 nM in the next ∼25 min. A pronounced drop in cellular ATP levels and a rapid increase in intracellular Na+ concentrations in the first 20 min of MI excluded Ca2+ efftux in the second phase via plasma membrane ATPases or Na+/Ca2+ exchangers (NCE). Mitochondrial rhod 2 intensities increased to 434 ± 46% of the control value during MI, indicating that mitochondria sequester Ca2+ during MI. The mitochondrial potential (Δψm) was lost in 20 min of MI, excluding mitochondrial Ca2+ uptake via the ΔΨm-dependent mitochondrial Ca2+ uniporter after 20 min of MI. Under Na+-free conditions, or when CGP-37157, a specific inhibitor of the mitochondrial NCE, was used, no drop in [Ca2+]i was seen during MI, whereas the MI-induced increase in mitochondrial rhod 2 fluorescence was strongly reduced. To our knowledge, this study is the first to report that in metabolically inhibited renal epithelial cells mitochondria take up Ca2+ via the NCE acting in the reverse mode. [ABSTRACT FROM AUTHOR]