Figure 5

Fluorescent approach to the measurement of intracellular Ca++ ions is represented in (5A). The IC50 value at 24 hrs, as determined by MTT assay, was 10 nM taxotere. The ratio of fluorescent intensity for the two-excitation wavelengths (345 and 390 nm) was plotted against duration of drug exposure. Increases in ratio imply increases in [Ca]_i. Open circles show the mean value (with SE bars) as a function of drug exposure and indicate [Ca]_i elevation from the taxotere. Some of the wells were then treated with thapsigargin, which caused the release of Ca⁺⁺ from intracellular stores (e.g., endoplasmic reticulum). Filled triangles show the mean ratio for each set of wells 20 mins after addition of thapsigargin to each well. Likewise, some of the wells were then treated with ionomycin, a Ca⁺⁺ ionophore (open squares). Inverted triangle is ratio for dye free cells. These values become very small at high, saturating [Ca]_i values and inaccuracies in the control zero value can produce errors. (5B) represents approach to the measurement of Ca⁺⁺. All 3 human prostate cancer lines readily took up sodium green Na+ sensing fluorescent dye. Data is displayed as fluorescence intensity (vertical axis) versus forward scatter (horizontal axis). Comparing control versus treated (VP-16 against DU145 for 24 hrs), there was a clear second population of cells with increased fluorescence and decreased size. The second population appears here at the upper left of the main population. The two populations remained after gating out the high propidium iodide cells (presumably necrotic), yet have distinctly different propidium iodide intensity levels (i.e., there are 3 populations on a propidium iodide plot).