Fura-2-based Ca2+ Imaging in MIA PaCa-2 Cells

MIA PaCa-2 cells were loaded with 5 μm of fura-2-AM for 40 min at room temperature and imaged using a Nikon TE2000S microscope with ×40 oil immersion SFluor objective lens, CoolSNAP HQ CCD camera (Photometrics, Tucson, AZ) and Cairn monochromator (Cairn Research, Kent, UK), controlled by MetaFluor imaging software (Molecular Devices, Downington, PA). Background-subtracted 340- and 380-nm fluorescence images were captured with 50-ms exposure and 5 × 5 binning every 5 s, and emitted light was separated using a 400-nm dichroic with 505LP filter. The fura-2 fluorescence was calibrated into “estimated” [Ca²⁺]_i as previously described.^{10 (link),11 (link)}