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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. ATP dose response curves in the absence of probenecid.                        B. ATP dose response curves in the presence of 0.5 mM probenecid.                        C. ATP dose response curves in the presence of 1 mM probenecid.                        D. ATP dose response curves in the presence of 2 mM probenecid.
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Figure 5: A. ATP dose response curves in the absence of probenecid. B. ATP dose response curves in the presence of 0.5 mM probenecid. C. ATP dose response curves in the presence of 1 mM probenecid. D. ATP dose response curves in the presence of 2 mM probenecid.

Mentions: Because of high organic anion transporter activity in CHO cells, fluorescent calcium dyes are poorly retained in the absence probenecid. In a standard protocol for CHO cell calcium flux assays using Fluo-3, Fluo-4 and other commercial kits, 2.5 mM probenecid is required to achieve the optimal signal. Moreover, when CHO cells are transfected with GPCR genes, overexpression of the GPCR can have a negative impact on the host cells’ ability to tolerate prolonged incubation with probenecid. The BD™ PBX Calcium Assay Kit was specially designed for assays that are sensitive to probenecid, and the special formulation of the PBX kit helps increase dye retention in cells. To assess the performance, a probenecid titration assay (0 to 2 mM probenecid) was performed and measured on a FlexStation using the first generation BD calcium assay kit, BD PBX calcium assay kit and a Calcium 3 kit purchased from Molecular Devices. The data indicates that probenecid is not required for dye-loading CHO cells if the BD™ PBX Calcium Assay Kit is used (Fig. 5A) while 0.5 mM probenecid is sufficient for the BD™ Calcium Assay Kit (Fig. 5B). In contrast, the Calcium 3 kit requires at least 2 mM probenecid to exhibit a response (Fig. 5D).


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. ATP dose response curves in the absence of probenecid.                        B. ATP dose response curves in the presence of 0.5 mM probenecid.                        C. ATP dose response curves in the presence of 1 mM probenecid.                        D. ATP dose response curves in the presence of 2 mM probenecid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: A. ATP dose response curves in the absence of probenecid. B. ATP dose response curves in the presence of 0.5 mM probenecid. C. ATP dose response curves in the presence of 1 mM probenecid. D. ATP dose response curves in the presence of 2 mM probenecid.
Mentions: Because of high organic anion transporter activity in CHO cells, fluorescent calcium dyes are poorly retained in the absence probenecid. In a standard protocol for CHO cell calcium flux assays using Fluo-3, Fluo-4 and other commercial kits, 2.5 mM probenecid is required to achieve the optimal signal. Moreover, when CHO cells are transfected with GPCR genes, overexpression of the GPCR can have a negative impact on the host cells’ ability to tolerate prolonged incubation with probenecid. The BD™ PBX Calcium Assay Kit was specially designed for assays that are sensitive to probenecid, and the special formulation of the PBX kit helps increase dye retention in cells. To assess the performance, a probenecid titration assay (0 to 2 mM probenecid) was performed and measured on a FlexStation using the first generation BD calcium assay kit, BD PBX calcium assay kit and a Calcium 3 kit purchased from Molecular Devices. The data indicates that probenecid is not required for dye-loading CHO cells if the BD™ PBX Calcium Assay Kit is used (Fig. 5A) while 0.5 mM probenecid is sufficient for the BD™ Calcium Assay Kit (Fig. 5B). In contrast, the Calcium 3 kit requires at least 2 mM probenecid to exhibit a response (Fig. 5D).

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