Fura red

Isolated islets were loaded with 3μM Rhod-2 (Invitrogen), in imaging medium (125mM NaCl, 5.7mM KCl, 2.5mM CaCl₂, 1.2mM MgCl₂, 10mM Hepes, 2mM glucose, and 0.1% BSA, pH 7.4) for 45 minutes at room temperature, and were held in polymdimethylsiloxane PDMS microfluidic devices [85 (link)] maintained at 37°C. Rhod-2 fluorescence was imaged on a spinning disk confocal microscope (Marianas, 3I), excited at 561nm using an OPSL sapphire laser, with a 580-655nm band-pass filter for emission; or imaged on a confocal microscope (LSM780, Zeiss) excited at 561nm using a diode-pumped solid-state laser, with a 570-645nm band-pass selection for emission. A small sub-set of isolated islets were loaded with 4μM FuraRed (Invitrogen) for 90 minutes at room temperature, and imaged on a spinning disk confocal microscope, excited at 488nm using a diode-pumped solid-state laser, with a 580-655nm band-pass filter for emission. GFP fluorescence was excited at 488nm using a Ar+ laser line (LSM780) or diode-pumped solid-state laser (Marianas), with a 495-555nm band-pass selection for emission. Images were acquired 1/sec, 10 minutes after elevating glucose concentration (2-20mM). Microscope settings (integration time, scan time, gain, laser power) were constant for all images collected within the same day.

In order to measure acute opioid-induced changes in Ca²⁺ levels, cells were incubated for 30 min with Fura Red (Invitrogen), a ratiometric Ca²⁺ fluorescent indicator [78 (link),79 (link)] stimulated with 750 nM opioids and Ca²⁺ levels were monitored using sequential, i.e., dual-track time-lapse CLSM imaging. In the first track, the 488 nm line of the Ar/ArKr laser was used to excite eGFP and Fura Red. The eGFP signal was collected using the band pass 505-530 nm emission filter, and the Fura Red signal was collected using the long pass 680 nm emission filter. In response to changes in Ca²⁺ concentrations, the excitation wavelength of Fura Red shifts from 472 nm at low Ca²⁺ concentration to 436 nm at high Ca²⁺ concentration [78 (link)]. As a result, the intensity of the Fura Red fluorescence signal decreases when intracellular Ca²⁺ levels increase and increases as they fall again. In the second track, the 543 nm HeNe laser was used to excite Tomato, and fluorescence was collected using the band pass 580–620 nm emission filter. The images were collected every 30 s for 40 min (in some cases up to 80 min). The pixel dwell time was 51.2 µs, and the images were collected without averaging.

The laser scanning confocal microscope system Nikon A1-R was used to detect [Ca²⁺]_i transients. Samples were imaged using 20 × and 60 × objective lenses: Plan Apo 20x/NA 0.75 and 60x/NA 1.4 Oil. Open-source software ImageJ was used for analysis. Changes in [Ca²⁺]_i concentration in isolated glomeruli were estimated by fluorescent dyes: Fluo 4 (ex. 488 em. 520/20 nm; #20,190,588, Invitrogen, OR) and Fura Red (ex. 488 em. > 600 nm; #21,046, AAT Bioquest, Inc, CA).

A20 or A20 D1.3 B cells that had been transfected with siRNA, or treated with DMSO or LIMKi3, were washed twice with Hanks' Balanced Salt Solution (HBSS) containing 10 mM HEPES and resuspended to 10⁷ per mL before adding 2 μM Fura Red (Invitrogen, #F3021), 1 μM Fluo-4 (Invitrogen, #F14201), and 0.02% Pluronic F-127 (Invitrogen, #P3000MP). The cells were then incubated for 30 min at room temperature protected from light, washed with HBSS/10 mM HEPES/2% FCS, resuspended to 10⁷/mL, and incubated for an additional 20 min protected from light. Flow cytometry was performed using an LSRII-561 cytometer (Becton Dickinson Biosciences). For each sample, 1–3 × 10⁶ cells were pelleted and resuspended in 0.5 mL mHBS, with paired samples having similar number of cells. Samples were analyzed for 1 min to establish baseline values before adding either goat anti-mouse IgG (Jackson ImmunoResearch, #115-005-008, 20 μg/mL) for A20 B cells or goat anti-mouse IgM (Jackson ImmunoResearch, #115-005-020, 20 μg/mL) for A20 D1.3 B cells, and then analyzing the cells for an additional 5 min. Ionomycin (1 μM; Invitrogen, # I24222) was added to saturate the Ca²⁺-sensing dyes and the cells were analyzed for an additional 1 min. Data were analyzed using FlowJo software (Treestar Inc.), gating on single intact cells using forward and side scatter.