Xenon arc lamp

For fluorescence
imaging, a xenon
arc lamp equipped with a filter wheel and shutter (Sutter Instruments,
Novato, CA) containing 340 ± 5 and 380 ± 5 nm filters (Omega
Optical 340AF15 and 380AF15, Brattleboro, VT, USA) was focused onto
the islet chamber using a 10×, 0.5 NA microscope objective. Fluorescence
emission was collected with the same objective, passed through a dichroic
mirror and a 510 ± 84 nm emission filter (Semrock, Rochester,
NY, USA), and detected by a CCD camera (QImaging, Surrey, BC, Canada).
The images of all islets were collected every 20 s with 150 ms exposure
for each excitation wavelength. The ratio of fluorescence emission
at 510 nm after excitation at 340 and 380 nm was collected to calculate
free [Ca²⁺]_i using predetermined calibration
values by reported methods.^{63 (link)}To
quantify the period of calcium oscillations, we measured the time
between the beginnings of two consecutive oscillations was measured.
An oscillation was defined as when [Ca²⁺]_i elevated
above a threshold level until dropping below it. The threshold level
for each experiment was determined as the average free [Ca²⁺]_i during the nadirs of oscillations during the 20 mM
glucose period before treatment plus ten times the standard deviation.

Ratiometric calcium imaging experiments were conducted with the fluorescent indicator Fura-2AM (Fura-2, Thermo Fisher Scientific). Cells were loaded with 5 µM Fura-2 for 45 min and incubated at 37°C in a 5% CO₂ atmosphere. Recordings were performed in a low-volume chamber continuously perfused. Bath solution contained (in mM) 140 NaCl, 3 KCl, 2.4 CaCl₂, 1.3 MgCl₂, 10 HEPES and 10 glucose adjusted to pH 7.4 with NaOH. Fluorescence measurements were made with a Leica inverted microscope (Leica DMI 3000B, Leica Microsystems). Fura-2 was excited using a xenon arc lamp (Sutter Instruments) at 340 and 380 nm wavelengths, switching between both with a computer-controlled filter wheel (Lambda 10-2; Sutter Instruments), and the emitted fluorescence was long-pass filtered at 510 nm. Images were acquired using an ORCA ER CCD camera (Hamamatsu Photonics K.K.) at a frequency of 0.33 Hz and analysed with Metafluor software (Molecular Devices). Cytosolic Ca²⁺ increases are presented as the ratio of the emission intensities after excitation at 340 and 380 nm (F₃₄₀/F₃₈₀). Measurements in DRG neurons were performed at 33 ± 1°C. Unless indicated otherwise, calcium measurements in cells lines were performed at 24 ± 1°C, controlled by a homemade temperature feedback device.

Microfluidic devices were made from PDMS and fabricated using conventional soft lithography.²¹ The design used in this study was described in previous work.¹² Imaging of Ca²⁺ was performed as previously described.¹² Briefly, the microfluidic device was positioned on the stage of a Nikon Ti-S microscope equipped with a 10X, 0.5 NA objective (Nikon Instruments, Melville, NY). Excitation was achieved with a Xenon arc lamp equipped with an integrated shutter and filter wheel (Sutter Instruments, Novato, CA). Acquisition was performed on a charge coupled device (CCD) camera (Photometrics, Tucson, AZ). Nikon NIS Elements software was used to control the camera, shutter, and filter wheel. During imaging, excitation was performed sequentially at 340 ± 5.5 and 380 ± 5.5 nm (Chroma, Bellows Falls, VT). Emission was filtered through a 415 nm long pass dichroic mirror and a 510 ± 20 nm emission filter (Omega, Brattleboro, VT). The response of islets is given as a ratio of fluorescence emission generated from excitation at 340 and 380 nm (F340/F380). Imaging was performed every 30-s with 100-ms exposure per excitation channel.