Alterations in intracellular Ca²⁺ concentration are amongst the most rapid responses to a variety of stimuli in mammalian cells. In the nervous system in particular, responses occur within nanoseconds. A major challenge in intracellular Ca²⁺ analysis is to achieve measurements within this very fast time frame. To date, the dynamic intracellular Ca²⁺ concentration has been monitored by confocal microscopy, plate‐based assays, spectrofluorometry, and flow cytometry, although there are issues with the number of cells analyzed or gaps in recording due to the addition of compounds, with significant loss of detail of a rapid Ca²⁺ response. The new generation of flow cytometers (such as Accuri C6) resolves this problem by allowing the addition of test compounds with continuous monitoring of thousands of cells, providing a method for dynamic Ca²⁺ measurements. This system was tested with commonly used Ca²⁺ modulating agents in C6 glioma cells. Thapsigargin (TG), a blocker of Ca²⁺ uptake into the endoplasmic reticulum (ER), causes a significant increase in the intracellular calcium concentration via ER emptying followed by Ca²⁺ entry via store‐operated Ca²⁺ channels (SOCC). This well‐established pathway can be partially inhibited by 2‐aminoethoxydiphenyl borate (2‐APB), a blocker of SOCC. Both the increase with TG alone and the partial increase when coincubated with 2‐APB were observed with continuous recording along with calibration curves using an Accuri C6 flow cytometer. With these new cytometers, dynamic Ca²⁺ concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness. © 2010 International Society for Advancement of Cytometry

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