Bioimaging Systems

Figure 1. Response of CHOK1 Endogenous P2Y receptor to ATP. CHOK1 cells were plated overnight in 100µl culture medium on two 96-well Biocoat poly-D lysine coated plates. The next day, one plate of cells was dye-loaded with 100µl/well of BD RatioMax Calcium Assay Kit, and the other was loaded with Fura-2 AM after removing the medium. After 1 hour incubation at 37°C, cells loaded with Fura-2 were washed three times with HBSS-Hepes buffer. The same dilutions of ATP were added to both plates using a FlexStation (MDS). Dose response curves were generated by SoftMaxPro (MDS)

Figure 2. BD High-Performance Calcium Assay Kit
(Ex 485 nm, Em 525 nm) Kinetic curve of calcium response to Carbachol in presence and absence of NTS143 using BD High-Performance Calcium Assay Kit. NTS143 acted like an agonist.

Figure 3. BD RatioMax Calcium Assay Kit
(Ex 340 & 380 nm, Em 510 nm) Kinetic curve of calcium response to Carbachol in presence and absence of NTS143 using BD RatioMax Calcium Assay Kit. NTS143 performed as an antagonist.

Response of Muscarinic Receptor M1 to Fluorescent Antagonist N-(3-Triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (NTS143). HEK293 cells were plated overnight in 100µl culture medium on two 96-well Biocoat poly-D lysine coated plates. The next day, one plate was dye-loaded with 100µl of BD High-Performance Calcium Assay Kit and the other with 100µl of BD RatioMax Calcium Assay Kit at 37°C for one hour. Two additions, serial dilutions of NTS143 (visible light-excitable fluorescent compound) followed by 4µM carbachol (EC80) were made using a FlexStation (MDS).

Figure 4. Kinetic curves of calcium response to different concentrations of ATP on a BD Pathway 855.

Figure 5. Segmentation of the fluorescent signal into specific regions of interest (ROIs) by BD AttoVision™ software.

CHOK1 cells were plated overnight in 100µl culture medium on a 96-well BD Falcon™ Imaging Plate (Cat No. 353219). The next day, the plate was dye-loaded with 100µl of BD RatioMax Calcium Assay Kit and 2.5mM probenecid at 37°C for one hour.

Figure 6. Overlay of calcium response kinetic curves.

Figure 7. Data of a sample well showing emission signals excited at 340nm and 380nm and the ratio of the two emission signals

Response of Muscarinic Receptor to Carbachol. CHO-K1 cells stably transfected with muscarinic receptor were plated overnight in 3 µl culture medium on a 1536-well plate (Greiner). The next day cells were dye-loaded with 2.5 µl/well of BD RatioMax Calcium Assay Kit in the presence of 2.5 mM probenecid. After 1 hour incubation at 37°C, 1 µM carbachol or buffer was added by an FDSS 7000 and data was recorded simultaneously.