FLIPR Assay – FLlPR Calcium Assay

The FLIPR Calcium Assay or “FLIPR Assay” is a highly sensitive method for the quantitative determination of calcium levels in cells. The EC50 of the assay is 4 uM. The assay is suitable for all types of cell culture systems.

Performing The Test

To perform the test, we prepared a 384-well plate with growth medium and half of the wells incubated with the screen Quest(tm) Fluo-4 or Fluo-8 NW calcium assay kits. Incubation was performed at room temperature for one hour. The other half incubated with FLIPR 384, which contains Carbachol.

PPLS-HA-GPR88-Gaqi5-CHO cells

Stable PPLS-HA-GPR88 Gaqi5-CHO cells were seeded at 40,000 cells/well in a Costar 96-well plate. After incubation at 37 degC with 5% CO2, cells were stained with Calcium 5 dye. After incubation, cells were added to final concentrations indicated on the kit.

HBSS with additional 24 mM (5X) calcium

Using a Fluorometric Imaging Plate Reader (FIPR), we measured the amount of calcium in each cell well using the FLIPR Calcium Assay Kit. We first detached HEK-3.x cells from flasks using trypsin/EDTA, resuspended them in Leibovitz’s L-15 medium containing 1% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Then we plated the cells in 90 mL Corning 96-well microplates and measured their fluorescence over this time.

In-tip dilution of FLIPR Calcium 6 Assay Kit

In-tip dilution of FLIR (Fluorescence-Activated Protein Receptor) Calcium 6 Assay Kit is a reagent used to detect agonists, antagonists, and allosteric modulators. The assay is performed by adding a small amount of the screening compound to the solution. This small amount causes a calcium flux response that corresponds to ten to ninety percent of the maximal response. To test for antagonism, the maximum concentration of the known agonist is added to the solution.

Z’-factor of FLIPR Calcium Assay

The Z’-factor of FLIPR Calcium assay is important for accurate quantification of calcium. The assay measures calcium flux response from a cell culture. Generally, an agonist has a high Z’-factor, while an antagonist has a low Z’-factor. Nevertheless, the z’-factor of FLIPR Calcium Assay must be higher than 0.8 for optimal detection.

No-wash dyes used in FLIPR Calcium Assay

Fluorescent calcium indicators have been used in the laboratory for decades, but only two recent generations are no-wash dyes: FLIPR Calcium 6 and Cal-520 AM. These dyes retain their fluorescence signal after washing but the signal from FLIPR Calcium 6 is larger than from Cal-520 AM, indicating a favourable use of no-wash dyes. Neither of these dyes is toxic to cells, but they have varying degrees of sensitivity.