Alterations in intracellular Ca 21 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 Ca 21 analysis is to achieve measurements within this very fast time frame. To date, the dynamic intracellular Ca 21 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 Ca 21 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 Ca 21 measurements. This system was tested with commonly used Ca 21 modulating agents in C6 glioma cells. Thapsigargin (TG), a blocker of Ca 21 uptake into the endoplasmic reticulum (ER), causes a significant increase in the intracellular calcium concentration via ER emptying followed by Ca 21 entry via store-operated Ca 21 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 Ca 21 concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness. ' AN alteration in intracellular calcium concentration is one of the most common second messenger responses now known in mammalian cells. These responses have been shown to play a critical role in long established mechanisms such as action potential generation in electrically excitable cells (1,2) and have been implicated in a wide variety of intracellular signaling and regulatory pathways, including initiation of apoptosis (3), alteration of cell surface protein expression (4), and response to mechanical stress on the plasma membrane (5). The vast majority of changes in intracellular calcium is extremely rapid and occurs within a nanosecond timescale. Therefore, if the full implications of a change in the intracellular calcium concentration are to be understood, then a protocol for extremely rapid and sensitive intracellular calcium determination is required.Methods currently in use for intracellular calcium determination include confocal microscopy, plate-based assays, spectrofluorometry, and flow cytometry. Although confocal microscopy examines in great detail the intracellular calcium dynamics over time, the number of cells examined per field is low, and therefore, much of the response throughout a population of cells may be unrecorded. Plate-based assays overcome this issue by recording the response of the entire population...

These data suggest that the bystander signal produced in a multicellular environment induces complex changes in the ITCM-treated culture, and that these changes are reflective of a coordinated response to maintain integrity throughout the tissue.

These data suggest, for these cell lines at least, that in the case of cell lines capable of responding to the bystander signal, it is the signal produced by the irradiated cell that determines the magnitude of the bystander effect.

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca2+ ATP pump inhibitor, thapsigargin (10 microM) produced a time-dependent increase in the Vmax for D-[3H]aspartate transport that reached a maximum at 15 min (143% of control; P<0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the Vmax of sodium-dependent D-[3H]aspartate transport (190% of control; P<0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in D-[3H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 microM) or SKF 96365 (10 microM) caused a net inhibition of D-[3H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in D-[3H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.

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