A flow-cytometric method for continuous measurement of intracellular Ca(2+) concentration.

Alterations in intracellular Ca(2+) 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(2+) analysis is to achieve measurements within this very fast time frame. To date, the dynamic intracellular Ca(2+) 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(2+) 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(2+) measurements. This system was tested with commonly used Ca(2+) modulating agents in C6 glioma cells. Thapsigargin (TG), a blocker of Ca(2+) uptake into the endoplasmic reticulum (ER), causes a significant increase in the intracellular calcium concentration via ER emptying followed by Ca(2+) entry via store-operated Ca(2+) 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(2+) concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness.

Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx.

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.

Bystander signal production and response are independent processes which are cell line dependent.

PURPOSE: Radiation-induced bystander effects are now an established phenomenon seen in numerous models; however it is not known whether the magnitude of the bystander effect is determined by the signal produced by the irradiated cells or the response of the exposed cells. The aim of this investigation is to determine the importance of the bystander signal versus the bystander response in three different cell line models. METHODS: A matrix design experiment using cell lines, HPV-G, CHO-K1 and E89 (glucose 6-phosphate dehydrogenase (G6DP) null) was set up to produce irradiated cell conditioned medium (ICCM), which was used to treat all cell lines. RESULTS: For HPV-G and CHO-K1 lines, the response to autologous ICCM was significantly different to that when treated with ICCM generated from another line. These lines displayed no response to E89 ICCM, nor did E89 cells show a significant response to any ICCM, suggesting that G6DP plays a key role in the bystander effect. CONCLUSION: 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.