Fura-2 QBTカルシウムキット

Figure 1: On the FlexStation® 3 Microplate Reader, carbachol was used to stimulate the muscarinic M1 receptor on CHO M1 cells to generate concentration response curves (CRC), comparing Fura-2 QBT Dye to the BD Kit and the traditional Fura-2 wash protocol. EC50 values were similar for all methods, and were within range of published values.

Figure 2: On the FlexStation 3 Microplate Reader, 50 nM carbachol was used as agonist challenge versus an antagonist CRC of atropine. This experiment Fura-2 QBT Calcium Kit provided the largest signal window and most robust Z-factors at EC80.

Figure 3: Histamine was used to stimulate the endogenous histamine receptor on HeLa cells. Assay was measured using the FLIPR Tetra Instrument with UV LEDs. Generated concentration response curves (CRC) comparing Fura-2 QBT Dye to the BD Ratiometric Calcium Kit and traditional Fura-2 wash protocol. EC50 values were within a half log while the Z-factor at EC80 was the largest with the Fura-2 QBT Calcium Kit.

Figure 4: Assay was measured using the FLIPR Tetra Instrument with UV LEDs and used similar experimental setup and system settings in Figure 3. 40 nM histamine was used as agonist challenge versus an antagonist CRC of pyrilamine.

Figure 1: Fura-2 QBT™ Calcium Dye is excited at 340nm and 380 nm, with emission at 510 nm. Upon calcium flux, one can measure changes in intracellular calcium concentration.

Figure 2: Calcium-bound Fura-2 is excited at 340/510 nm and the unbound form at 380/510 nm. Upon calcium-flux, the cytoplasm’s calcium concentration increases, causing the fluorescence intensity at 340/510 nm to increase proportionately. In contrast, the 380/510 nm intensity declines in synchronization with the diminishing concentration of unbound dye. By ratioing fluorescence intensities produced by excitation at two wavelengths, artifacts associated with uneven dye distribution and cell number variation are minimized because they effect both measurements equally.

Figure 3: Fura-2 QBT Calcium Kit utilizes Fura-2, AM a calcium sensitive dye that is absorbed into the cell’s cytoplasm during incubation, where lipophilic blocking groups are cleaved resulting in a negatively charged fluorescent dye that stays within the cell. Upon target activation, intracellular calcium is released, binding with the dye, thereby increasing fluorescence intensity. This kit utilizes an extracellular masking technology to block background fluorescence and increase your assay’s signal window, and decrease assay steps.