A Dual-Color Reporter for Insulin+ Beta Cells Figure 4. INS and GCG transcript expression in FACS-sorted GFP+ and mCherry+ cells using q-RT-PCR. (a) After FACS analysis in stable HIT-T15 cell population to select GFP+ or mCherry+ cells, q-RT-PCRs were performed in GFP+ and mCherry+ cells. (b, c) q-RT-PCR data confirmed that the green and red cells were insulin- and glucagon-producing, respectively. (d, e) The green and red cells were analyzed for expression levels of Pdx1 (d) and Arx (e) genes by q-RT-PCR. The expression levels of INS, GCG, Pdx1, and Arx transcripts were normalized to the housekeeping genes, GAPDH and/or Cyclophilin (‘‘MNE’’). The error bars represent standard error (SEM) of the mean (N = 3 for each experiment). doi:10.1371/journal.pone.0035521.g004 compared to red cells [74% (21 out of 30) versus 28% (7 out of 25)], as inferred from changes in membrane capacitance following trains of eight depolarizing pulses (from 280 to 0 mV, 100 ms duration; Fig. 5e & f). Adding Cd2+ blocked changes in membrane capacitance, as would be expected for insulin secretion, which is known to be predominantly Ca2+-dependent (Fig. 5d). The main voltage-gated Ca2+ channels controlling insulin secretion in beta-cells are L-type channels that can be blocked by nitrendipine [23]. Addition of 20 mM nitrendipine only partially blocked the Ca2+ current (,50%) but almost totally eliminated the capacitance increase in green cells (Fig. 5f). This suggests that more than one type of Ca2+ channel is present, but that L-type Ca2+ channels gate the Ca2+ entry that is coupled to exocytosis. There was a greater variability in the amplitude of calcium currents and capacitance changes in red cells compared to green cells. One characteristic of beta-cells is that intracellular Ca2+ levels ([Ca2+]i) rise in response to increased extracellular glucose [24,25]. We used ratiometric imaging of the membrane-permeant calcium indicator dye, Fura2-AM (Fura2 acetoxymethyl ester [26]), in cells exposed to either basal (3 mM) or stimulatory (10–14 mM) levels of glucose. The fluorescence ratio (350 nm/ Figure 5. Electrophysiological differences between GFP+ and mCherry+ cells. (a) GFP+ cells exhibited significantly greater calcium currents compared to mCherry+ cells (Mann Whitney, P,0.0001, n = 19–22). (b) Example current traces from GFP+ cells (top panel) and mCherry+ cells (bottom panel). Scale bar: 100 pA, 10 ms. (c) Average current-voltage plot showing greater calcium influx in GFP+ cells (closed circles) compared to mCherry+ cells (open circles). (d) Cadmium (Cd2+, 30 mM) blocked inward current in a GFP+ cell. (e) Depolarizing trains of 8 pulses (100 ms, 0 mV) induced an increase in capacitance (corresponding to an increase in exocytosis) from most of GFP+ cells (21 out of 30). Only 7 out of 25 cells mCherry+ cells underwent an increase in capacitance when stimulated by 8-pulse depolarizing trains. (f) Capacitance recording from the same GFP+ cells with and without 20 mM Nitrendipine. doi:10.1371/journal.pone.0035521.g005 PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35521