Ratiometric Ca⁺²measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer

Abstract

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are "gold standard" because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the "mix & measure" assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca+2 measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compared the IC50 values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca+2 mobilization assays can be an alternate to radioligand binding assays.

Dental enamel cells express functional SOCE channels

“…Finally, cells were dispensed into 96-well black clear bottom plates. Fura-2 emission was detected at 510 nm after excitation at 340 nm and 380 nm using a FlexStation3 plate reader (Molecular Devices, USA) 46 . The ratio (R) of Fura-2 emission following excitation at 340 nm (F340) and 380 nm (F380) were calculated for each time point.…”

Epigenetic assays for chemical biology and drug discovery

“…In the case of the neurotransmitter targets, these assays are considered to be the gold standard assay format as they are label-free, highly sensitive and are not prone to interference in a manner that the other optical methods are susceptible [109]. An advancement to these assays is the no-wash scintillation proximity assay (SPA) which makes use of beads embedded with scintillant that can bind the target of interest and give a signal [110–112].…”

Store-operated Ca2+ Entry Modulates the Expression of Enamel Genes

“…After loading, cells were washed and dispensed in 96-well black glass-bottom plates (Molecular Devices). Average fluorescence was measured by FlexStation III (Molecular Devices, Sunnyvale, CA, USA), a 96-well fluorescence spectrometer, at 340-and 380nm excitation wavelengths and a 510-nm emission wavelength as previously described (Gupta et al 2009) at 25 °C. Data acquisition was performed using SoftMax Pro (Molecular Devices).…”