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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.


Related in: MedlinePlus

A. Dose response curves of ATP in HEK 293 cells in the absence of probenecid in 384-well format. Signal to background are 3.9 and 4.6, EC50 values are 1.1 µM and 1.5 µM using the first and second generation kits, respectively.                        B. Dose response curve of ATP in CHO-K1 cells in the presence of 2.5 mM probenecid in 96-well format. Signal to background are 5.0 and 5.5, EC50 values are 67 nM and 58 nM using the first and second generation kits, respectively.
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Figure 2: A. Dose response curves of ATP in HEK 293 cells in the absence of probenecid in 384-well format. Signal to background are 3.9 and 4.6, EC50 values are 1.1 µM and 1.5 µM using the first and second generation kits, respectively. B. Dose response curve of ATP in CHO-K1 cells in the presence of 2.5 mM probenecid in 96-well format. Signal to background are 5.0 and 5.5, EC50 values are 67 nM and 58 nM using the first and second generation kits, respectively.

Mentions: To assess the performance of the BD calcium assay kits, the endogenous P2Y receptors in HEK293 [8] and CHO-K1 [9] cells were compared side by side using identical assay conditions. To maintain a true no-wash assay, culture medium was not removed before dye-loading. HEK 293 cells in general do not require probenecid for dye-loading, and therefore probenecid was not included in the dye-loading solution (Fig. 2A). However, probenecid was added to prevent dye leakage when CHO cells were assayed (Fig. 2B). The fluorescent signal change at different concentrations of the ligand ATP was calculated and plotted. As shown in Fig. (2), the second generation kit (BD™ High Performance Calcium Assay Kit) exhibited improved signal to background in both HEK293 and CHO cells at the highest ATP concentrations, while the EC50 values remained similar.


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. Dose response curves of ATP in HEK 293 cells in the absence of probenecid in 384-well format. Signal to background are 3.9 and 4.6, EC50 values are 1.1 µM and 1.5 µM using the first and second generation kits, respectively.                        B. Dose response curve of ATP in CHO-K1 cells in the presence of 2.5 mM probenecid in 96-well format. Signal to background are 5.0 and 5.5, EC50 values are 67 nM and 58 nM using the first and second generation kits, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: A. Dose response curves of ATP in HEK 293 cells in the absence of probenecid in 384-well format. Signal to background are 3.9 and 4.6, EC50 values are 1.1 µM and 1.5 µM using the first and second generation kits, respectively. B. Dose response curve of ATP in CHO-K1 cells in the presence of 2.5 mM probenecid in 96-well format. Signal to background are 5.0 and 5.5, EC50 values are 67 nM and 58 nM using the first and second generation kits, respectively.
Mentions: To assess the performance of the BD calcium assay kits, the endogenous P2Y receptors in HEK293 [8] and CHO-K1 [9] cells were compared side by side using identical assay conditions. To maintain a true no-wash assay, culture medium was not removed before dye-loading. HEK 293 cells in general do not require probenecid for dye-loading, and therefore probenecid was not included in the dye-loading solution (Fig. 2A). However, probenecid was added to prevent dye leakage when CHO cells were assayed (Fig. 2B). The fluorescent signal change at different concentrations of the ligand ATP was calculated and plotted. As shown in Fig. (2), the second generation kit (BD™ High Performance Calcium Assay Kit) exhibited improved signal to background in both HEK293 and CHO cells at the highest ATP concentrations, while the EC50 values remained similar.

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.


Related in: MedlinePlus