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FLIPR Calcium 3 Assay Kit - An Optimal No-Wash Fluorescence Calcium Indicator Reagent for Non-Optimal Calcium Responses

neration of stable cell lines to confirm functional receptor expression from the plasmid construct, in assay development and validation, and in some cases for compound screening. The results presented highlight an attractive feature of the Calcium 3 no-wash reagent compared with a conventional dye loading and wash protocol for these non-optimal calcium responses. Use of Calcium 3 improved data quality as evidenced by an increase in signal dynamic range, improvement in well-to-well variability and virtual elimination of dilution artifact following compound addition.


METHODS
Cells
HEK-293 cells were used for the transient expression of the 5-HT2C (VSV isoform, tagged at the C-terminus with GFP), 5-HT1A, 5-HT6 or 5-HT7 receptor subtypes. The 5-HT2C receptor couples primarily via Gq to the activation of phospholipase C with subsequent elevation of intracellular calcium. Calcium signaling with the normally Gi-coupled 5-HT1A or Gs-coupled 5HT6 and 5-HT7 receptors was facilitated by transient co-expression with chimeric or promiscuous G-proteins. For transient expression, HEK cells seeded at an approximate density of 3x106 in 10 cm2 dishes were transfected using the Lipofectamine Plus reagent and 24 hours following transfection were re-plated into poly-D-lysine coated 96-well black wall clear bottom plates at a density of 60-80,000 cells per well and used the next day for FLIPR assays.

FLUO-4 FLIPR studies
5-HT stimulated calcium responses were measured using the FLIPR system. Growth medium was removed by aspiration and cells were washed twice with 150 l Hanks buffered saline solution supplemented with 20 mM HEPES and 2.5 mM probenecid (HBSS). Dye loading buffer comprised FLUO-4-AM at 2 M final concentration, 1% fetal bovine serum and 0.1% pluronic acid. After washing, c
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