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Improvements in live cell analysis of G protein coupled receptors using second generation BD calcium assay kits.

Li X, Llorente I, Brasch M - Curr Chem Genomics (2008)

Bottom Line: The kits use a proprietary formulation including a non-fluorescent calcium indicator that becomes activated inside the cell and shows increased fluorescence upon calcium binding.We have compared the next generation BD calcium assay kit product family to previous versions of the formulation, and to other commercially available homogeneous calcium assay kits.The improvements have enabled better performance on the cell lines and receptors that we have tested in all plate formats including 1536.

View Article: PubMed Central - PubMed

Affiliation: Bioimaging Systems, BD Biosciences, Rockville, Maryland, USA. xiao_li@bd.com

ABSTRACT
BD Calcium Assay Kits are designed for cell-based calcium mobilization high-throughput screening assays. The kits use a proprietary formulation including a non-fluorescent calcium indicator that becomes activated inside the cell and shows increased fluorescence upon calcium binding. The formulation includes a signal-enhancing reagent to maximize the signal over background in a homogeneous, no-wash assay format, based on a technology developed at BD. We have compared the next generation BD calcium assay kit product family to previous versions of the formulation, and to other commercially available homogeneous calcium assay kits. The improvements have enabled better performance on the cell lines and receptors that we have tested in all plate formats including 1536.

No MeSH data available.


A. CHO-M1 cells treated with 1 µM carbachol.                        B. Carbachol dose response curves plotted by Prism (GraphPad).
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Figure 4: A. CHO-M1 cells treated with 1 µM carbachol. B. Carbachol dose response curves plotted by Prism (GraphPad).

Mentions: Assay miniaturization increases the speed and efficiency of screening and lead discovery. However, miniaturization of cell-based assays to a 1536-well format is challenging, due in part to difficulties in cell washing and obtaining appropriate plating densities. The impact of cell washing can be reduced by employing homogeneous true no-wash assay formats. We therefore evaluated the performance of both BD calcium formulations on CHO cells stably expressing the rat muscarinic receptor M1 (CHO-M1) in a 1536-well format using an FDSS 7000. Similar to the results obtained in 96 and 384-well formats above, the BD™ High Performance Calcium Assay Kit provided higher response to carbachol, an agonist of muscarinic receptor, than the first generation kit (Fig. 4A). Both kits delivered robust signals, and there was no significant difference in EC50 and Z’ values [10] (Fig. 4B).


Improvements in live cell analysis of G protein coupled receptors using second generation BD calcium assay kits.

Li X, Llorente I, Brasch M - Curr Chem Genomics (2008)

A. CHO-M1 cells treated with 1 µM carbachol.                        B. Carbachol dose response curves plotted by Prism (GraphPad).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803433&req=5

Figure 4: A. CHO-M1 cells treated with 1 µM carbachol. B. Carbachol dose response curves plotted by Prism (GraphPad).
Mentions: Assay miniaturization increases the speed and efficiency of screening and lead discovery. However, miniaturization of cell-based assays to a 1536-well format is challenging, due in part to difficulties in cell washing and obtaining appropriate plating densities. The impact of cell washing can be reduced by employing homogeneous true no-wash assay formats. We therefore evaluated the performance of both BD calcium formulations on CHO cells stably expressing the rat muscarinic receptor M1 (CHO-M1) in a 1536-well format using an FDSS 7000. Similar to the results obtained in 96 and 384-well formats above, the BD™ High Performance Calcium Assay Kit provided higher response to carbachol, an agonist of muscarinic receptor, than the first generation kit (Fig. 4A). Both kits delivered robust signals, and there was no significant difference in EC50 and Z’ values [10] (Fig. 4B).

Bottom Line: The kits use a proprietary formulation including a non-fluorescent calcium indicator that becomes activated inside the cell and shows increased fluorescence upon calcium binding.We have compared the next generation BD calcium assay kit product family to previous versions of the formulation, and to other commercially available homogeneous calcium assay kits.The improvements have enabled better performance on the cell lines and receptors that we have tested in all plate formats including 1536.

View Article: PubMed Central - PubMed

Affiliation: Bioimaging Systems, BD Biosciences, Rockville, Maryland, USA. xiao_li@bd.com

ABSTRACT
BD Calcium Assay Kits are designed for cell-based calcium mobilization high-throughput screening assays. The kits use a proprietary formulation including a non-fluorescent calcium indicator that becomes activated inside the cell and shows increased fluorescence upon calcium binding. The formulation includes a signal-enhancing reagent to maximize the signal over background in a homogeneous, no-wash assay format, based on a technology developed at BD. We have compared the next generation BD calcium assay kit product family to previous versions of the formulation, and to other commercially available homogeneous calcium assay kits. The improvements have enabled better performance on the cell lines and receptors that we have tested in all plate formats including 1536.

No MeSH data available.