Three additional samples of live LLC-Pk1 cells were treated with fluo-4. Control group cells were submerged in HBS buffer, while ATP and bradykinin experimental groups were submerged in calcium free HBS buffer, eliminating significant extracellular calcium. Cells were then treated with respective stimuli, and sub sequentially treated with ionomycin. Time lapse images were taken at 10X magnification for 270 s (1 frame every 2s) to capture the cellular response to the stimuli and ionomycin via fluorescent activity (Figure 4). Fluorescence intensity versus time data reveal patterns of fluorescence intensity (and therefore, cytosolic calcium level) fluctuation based on specific stimuli introduced to the extracellular space (Figure 2). Control sample (ATP/ionomycin treated cells in HBS) exhibited an oscillating fluctuation in fluorescence intensity over time (Figure 3A) and did not demonstrate a relative fluo-4 intensity increase after ionomycin treatment (Table 1). Bradykinin experimental cells demonstrated no response over time after being treated with stimuli, whereas a majority of ATP experimental cells exhibited a response (0/101, 152/154, respectively, Table 2). However, after ionomycin introduction, both bradykinin and ATP experimental cells experienced an increase in fluorescence intensity (800, 500, respectively, Table 2) for the remainder of the time lapse imaging (32s, Table 2). Differences in control and ATP experimental groups are noted in the duration of initial calcium spike (12 s, 8 s, respectively, Table 2), avg. time to first peak of cells responding to initial stimulus (42 s, 32 s, respectively, Table 2) and increase in intensity at peak of response to the stimuli (240, 850, respectively, Table 2).
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