Dage mti iccd camera

[Ca²⁺]_i was measured as previously described[33 (link)]. Briefly, serum-starved Caco-2 cell monolayers on glass-bottom microwell dishes (MatTek; Ashland, MA) were incubated with Fura-2AM (10 µM) in 0.5% pluronic acid for 30 min. Fura-2-loaded cells were alternately excited at 340 or 380 nm using a PC-driven hyper-switch (Ionoptix; MA, USA). Ratios were collected every second at 510 nm using a Dage MTI iCCD camera and Ionwizard software (Ionoptix). [Ca²⁺]_i was calculated using the following equation: [Ca²⁺]_i = K_d (R − R_min) (S_f2) / (R_max − R) (S_b2), where R is the 340/380 nm ratio, R_min and R_max are the minimum and maximum ratios determined in Ca²⁺-free and saturated Ca²⁺ solutions, respectively, S_f2/S_b2 is the Ca²⁺ free/Ca²⁺-replete ratio of emissions at 380 nm excitation, and K_d is the dissociation constant for Fura-2. R_min, R_max, S_f2, and S_b2 were determined by increasing the Ca²⁺ permeability of Caco-2 cells with ionomycin (10 µM), and perfusing cells with a high-Ca²⁺ (10 mM) or Ca²⁺-free (10 mM EGTA) solution. The in situ apparent dissociation constant (K_d) for Fura-2 used in this study was 224 nM. Eight to ten cells in each monolayer were analyzed simultaneously, and the experiments were repeated in 3–5 monolayers.