The FLIPR® System

Intracellular Calcium Assays

 
The most extensive use of the FLIPR® system to date has been in measuring intracellular calcium. In experiments using more than 20 mammalian cell lines, both adherent and non-adherent, the FLIPR system has proven to be very sensitive in detecting and quantifying changes in calcium. The most common calcium indicators used in the FLIPR system are the FLIPR Calcium Flux Assay Kit, the FLIPR Calcium 3 Assay Kit, Fluo-3, Fluo-4, and Calcium Green-1. All of these indicators excite in the visible wavelength range.

The main window of the FLIPR system control software is shown in Figure 1. All 96 wells are displayed simultaneously for real-time experiment monitoring. The data shown were generated using primary cultured neurons. Columns A-F were stimulated with 60 mM extracellular potassium, activating a voltage-gated calcium channel. Columns G and H were negative controls. The well-to-well data fidelity provided by the FLIPR system has allowed most screens to be run as single data points, greatly enhancing the effective throughput of the instrument.

		Figure 1. The main window of the FLIPR I system control software.

The results of a FLIPR system calcium experiment are shown in Figure 2. In this experiment, activation of the nociceptin receptor was linked to calcium via the introduction of the promiscuous G_α16 protein. As the data show, cells without the G-protein link did not effectively couple to calcium, whereas those that were transfected with the protein showed strong coupling. Each treatment was performed in duplicate, and data updates were taken every second. The data from the duplicates illustrate the data fidelity obtained using the FLIPR system.

		Figure 2. The effect of increasing concentrations of nociceptin on CHO cells.

Non-adherent cells, such as the human monocytic leukemia cell line THP-1, can also be used on the FLIPR system. The cells were activated with increasing concentrations of UTP, causing a response from an endogenous purinergic receptor. The FLIPR system has the ability to perform two different fluid additions (either from 96-well microplates or a common reservoir) during any one experiment.

		Figure 3. The effect of increasing concentrations of UTP on THP-1 cells.

intracellular calcium on FLIPR systems

CHO cells transfected with an M1 muscarinic receptor were seeded overnight onto a 384-well microplate and loaded with 4 µM Fluo-3. Triplicate wells were pre-exposed to increasing concentrations of the M1-selective antagonist pirenzepine from 0-10 µM (horizontal). Individual wells were then exposed to increasing concentrations of the agonist carbachol from 1 nM-10 mM (vertical). Using such data, it is possible to construct concentration-response curves for the response to carbachol in the presence of the antagonist, and thereby obtain pKb values for the antagonist.

		Figure 4. The effect of increasing concentrations of pirenzipine and carbachol on CHO cells.