Plan apochromat 20x 0.8 na lens

Ca²⁺ oscillation experiments were conducted in a microfluidic device (whole islets) or glass bottom micro-well dishes (dispersed β-cells) at 37°C and 5% CO₂. Intracellular calcium concentration ([Ca²⁺]_i) oscillation frequency was measured pre- and post-treatment with KP, GLP-1, gallein, or mSIRK individually and in combination, as previously described [17] . Intact islets were labeled with Fluo4-AM (4 µM; Invitrogen) in imaging buffer with 2 mM glucose for 30–45 minutes prior to data collection. Oscillations in Fluo4 fluorescence over the whole islet area were detected by excitation at 488 nm on a LSM 5Live microscope (Carl Zeiss) with a Plan-Apochromat 20x/0.8 NA lens. Images were collected at 1 frame every 2 seconds to measure the fast oscillations in [Ca²⁺]_i generated by changes in ion channel conductances (∼25 sec). Cells were imaged at 10 mM glucose for ∼5–10 min to allow sufficient time for synchronous oscillations to appear; the GPCR ligand and/or G_βγ modulator of interest then was added and oscillations were continuously recorded for another ∼10 min. Data were normalized to the untreated control frequency or amplitude for each islet prior to addition of a GPCR ligand and/or G_βγ modulator.