A Dual-Color Reporter for Insulin+ Beta Cells 380 nm) increased when a green cell was exposed to the stimulatory level of glucose (compare the green line in the left and right panels of Fig. 6a), indicating an increase in [Ca2+]i. The other colored lines in Figure 6a represent non-fluorescent cells, which do not exhibit a fluorometric ratio change under the same conditions. Ratiometric analysis of red cells did not show any change when shifted from the basal to the stimulatory glucose concentration (Fig. 6b). We also performed ratiometric imaging on INS-1 823/13 cells and observed increased [Ca2+]i in both basal and stimulating glucose concentrations (Fig. 6c). INS-1 823/13 cells, which retain insulin over-expression under a ubiquitous promoter (in contrast to HIT-T15 cells), thus appear to have spontaneous [Ca2+]i oscillations that are independent of glucose concentration. Figure 6. Intracellular Ca2+ concentration, [Ca2+]i changes associated with glucose stimulation in different fluorescent cells. (a) Glucose responsiveness of GFP+ stable HIT-T15 cells. The green line represents a GFP+ cell in basal (3 mM, left panel) and stimulatory (14 mM, right panel) levels of glucose. The other lines (red, blue, and orange) represent non-fluorescent cells. (b) No glucose responsiveness of mCherry+ stable HIT- T15 cells was observed. (c) Fluorescence traces from INS-1 cells. Each color represents a different cell, indicating spontaneous Ca2+ oscillations arising from action potentials. doi:10.1371/journal.pone.0035521.g006 PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35521