Hcx pl fluotar l 20x 0.40 na corr

Imaging experiments described in this study were performed as follows unless specifically noted. Epifluorescence imaging experiments were performed on a Leica DMI8 microscope (Semrock bandpass filter: GFPratio ex/em: FF01-391-23/FF01-520-35, GFP ex/em: FF01-474-27/FF01-520-35, RFP ex/em:FF01-554-23 or FF01-578-21/FF01-600-37, Far-red ex/em: FF01-635-18/FF01-680-42) controlled by MetaMorph Imaging software, using sCMOS camera (Photometrics Prime95B) and 20x magnification lens (Leica HCX PL FLUOTAR L 20x/0.40 NA CORR) or 10× objective (Leica HC PL FLUOTAR L 10x/0.32 NA) Confocal imaging experiments were performed on Leica SP8 confocal microscope from Imaging Core of Institute of Stem Cell and Regenerative Medicine. Cells were imaged in live cell imaging solution with 10mM glucose (LCIS+, Gibco, A14291DJ). Image analysis methods are described below.

On the day of imaging, ~24–36 hours post-transfection, cells were washed once with imaging solution, then replaced with imaging solution (Tyrode’s pH = 7.33; 125mM NaCl, 2mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 30 mM Dextrose, 25 mM HEPES (triple supplemented with 1% Glutamax (Gibco; 35050-1), 1% Sodium Pyruvate (Gibco; 11360-070), and 1% MEM Non-Essential Amino Acids (Gibco; 11140-050)). Powdered Potassium Chloride (Sigma; P9541-500G) was diluted in ddH₂O to a concentration of 2M. This solution was then diluted to 80mM in imaging solution (Tyrode’s pH = 7.33; 125mM NaCl, 2mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 30 mM Dextrose, 25 mM HEPES). During imaging, 1:1 volumes of KCl solution were hand-pipetted into the bath to bring the final KCl concentration to the desired concentration. Imaging was performed on a sCMOS camera (Photometrics Prime95B) on an epifluorescent microscope (Leica DMI8) using a 20X objective (Leica HCX PL FLUOTAR L 20x/0.40 NA CORR). A Lumencor Light Engine LED, and Semrock Filters (Excitation: FF01-474-27; Emission: FF01-620/35) were used for fluorescence imaging. Bulk fluorescence traces were acquired using FIJI imaging software with background subtraction (rolling = 50 stack) and hand-drawn ROIS. The baseline was defined as the first 30 measurements before KCl addition. Max $\mathrm{\Delta }\text{F}/{\text{F}}_0$ values were obtained using a custom Python script. Final traces were plotted in Prism9.

Imaging experiments described in this study were performed as follows unless specifically noted. Epifluorescence imaging experiments were performed on a Leica DMI8 microscope (Semrock bandpass filter: GFP ex/em: FF01–474-27/FF01-520-35, RFP ex/em:FF01-578-21/FF01-600-37) controlled by MetaMorph Imaging software, using a sCMOS camera (Photometrics Prime95B) and 20x magnification lens (Leica HCX PL FLUOTAR L 20x/0.40 NA CORR) or 10× objective (Leica HCX PL FLUOTAR L 10x/0.32 NA). Confocal imaging experiments were performed on a Leica SP8 confocal microscope from the Lynn and Mike Garvey Imaging Core at the Institute of Stem Cell and Regenerative Medicine. Cells were imaged in live cell imaging solution with 10mM glucose (LCIS+, Gibco, A14291DJ). Image analysis methods described below.