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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.


Kinetic data of CHO-K1 cells treated with 3 µM ATP.□ BD™ PBX Calcium Assay Kit.○ BD™ Calcium Assay Kit.△ Calcium 3 Kit.
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Figure 6: Kinetic data of CHO-K1 cells treated with 3 µM ATP.□ BD™ PBX Calcium Assay Kit.○ BD™ Calcium Assay Kit.△ Calcium 3 Kit.

Mentions: The performance of first generation BD kits and the Calcium 3 kit in the absence of probenecid was also compared side by side using a FLIPR 3. Endogenous P2Y receptor in CHO cells (Fig. 6) was used as an example. In CHO cells, the BD™ PBX Calcium Assay Kit was observed to generate 18,000 RFU of response to ATP and BD™ Calcium Assay Kit had 3000 RFU of response, while the Calcium 3 kit failed to deliver a positive signal. High fluorescent background in wells loaded with Calcium 3 kit indicated that the fluorescent calcium dyes were pumped out of cells in the absence of probenecid. When the agonist was added to the wells after dye-loading, the fluorescent molecules in extracellular environment were diluted and therefore a significant drop of fluorescent signal was observed. Because of poor dye-retention in cells, no fluorescent calcium flux signal could be detected by Calcium 3 kit.


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)

Kinetic data of CHO-K1 cells treated with 3 µM ATP.□ BD™ PBX Calcium Assay Kit.○ BD™ Calcium Assay Kit.△ Calcium 3 Kit.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Kinetic data of CHO-K1 cells treated with 3 µM ATP.□ BD™ PBX Calcium Assay Kit.○ BD™ Calcium Assay Kit.△ Calcium 3 Kit.
Mentions: The performance of first generation BD kits and the Calcium 3 kit in the absence of probenecid was also compared side by side using a FLIPR 3. Endogenous P2Y receptor in CHO cells (Fig. 6) was used as an example. In CHO cells, the BD™ PBX Calcium Assay Kit was observed to generate 18,000 RFU of response to ATP and BD™ Calcium Assay Kit had 3000 RFU of response, while the Calcium 3 kit failed to deliver a positive signal. High fluorescent background in wells loaded with Calcium 3 kit indicated that the fluorescent calcium dyes were pumped out of cells in the absence of probenecid. When the agonist was added to the wells after dye-loading, the fluorescent molecules in extracellular environment were diluted and therefore a significant drop of fluorescent signal was observed. Because of poor dye-retention in cells, no fluorescent calcium flux signal could be detected by Calcium 3 kit.

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.