The N-type calcium channel is a member of the voltage-sensitive calcium channel 
family and plays a major role in the regulation of neurotransmitter release in 
the central and peripheral nervous systems. Inhibition of the N-type calcium 
channel by intrathecal administration of the channel-specific blocker 
omega-conotoxin MVIIA (ziconotide) is efficacious in the treatment of severe 
chronic pain. While no orally active small molecules that block the N-type 
calcium channel are currently available, the discovery of such potentially 
valuable therapeutics would benefit from a reliable, high throughput assay. 
However, the assay of N-type calcium channel activity by measuring calcium 
influx using nonadherent cells in a high throughput fashion has not been 
achieved before, likely owing to a number of technical hurdles. For example, the 
measurement of calcium levels in nonadherent cells using conventional calcium 
indicators, such as Fluo-3 or Fluo-4, requires dyeloading the cells in 
suspension and subsequent removal of extracellular dye. This limits plate 
throughput and requires constant handling of the cells. To assay the N-type 
calcium channel activity using a nonadherent cell line in a high throughput 
manner, we investigated the application of no-wash calcium assay kits from 
Molecular Devices Corp. (Sunnyvale, CA): FLIPR Calcium, FLIPR Calcium Plus, and 
FLIPR Calcium 3. We show here that the FLIPR Calcium 3 assay kit can be used 
with nonadherent IMR-32 cells to measure potassium-evoked, omega-conotoxin 
MVIIA-reversible calcium flux with high throughput (15,000 data points/day), 
high quality (Z approximately 0.6), and minimal handling of the cells. Thus, 
this assay can be used to reliably and efficiently screen large compound 
libraries in the search for small molecule N-type calcium channel blockers.