Resuspension-Induced Ion Flux

Millar, David B.

doi:10.1006/abio.1997.2478

Cited by 1 publication

(1 citation statement)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although advertised as a "no-wash" product, 6 cell washing 1,4,[7][8][9] or media aspiration 2,10 steps are often part of the manufacturer's suggested protocols. However, using homogeneous calcium dye assay kits with washing or aspiration steps decreases their value by (1) including potential cell perturbation-causing calcium mobilization artifacts through washing (a similar mechanism demonstrated using centrifugation and resuspension 11 ), (2) not increasing screening throughput due to plate washing or media aspiration time and labor, and (3) in some kits, adding a proprietary quencher, a potential assay variable.…”

Section: Resultsmentioning

confidence: 99%

Identifying Nonselective Hits from a Homogeneous Calcium Assay Screen

Cassutt¹,

Orsini²,

Abousleiman³

et al. 2007

SLAS Discovery

View full text Add to dashboard Cite

The authors used a homogeneous calcium dye kit with a cell line transfected using a recombinant protein construct to screen a 50,000 compound library for G-protein coupled receptor (GPCR) agonists. Only 1 of the 365 primary hits activated Gq-coupled GPCRs, as shown using IP-ONE HTRF®. Furthermore, an agonist screen against the entire compound library and same heterologous cell line using AequoScreen™ technology generated no false positives and identified the same positive hit. Next, a multiplex assay composed of both Fluo-3 and Fura-2–loaded cells identified 1 false positive and the same true-positive hit out of the 365 primary hits. Finally, rescreening the 365 primary hits against the parental cell line loaded using the homogeneous calcium dye kit confirmed the specificity of the same true-positive hit only. In summary, the results suggest that AequoScreen™ technology, IP-ONE HTRF®, and multiplex assays are unique, orthogonal technologies to identify nonspecific hits.

show abstract