Download PDF

Introduction

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) activates Ca²⁺ entry across the plasma membrane in a variety of cell types, a process known as store-operated calcium entry (SOCE), and is the most common mode of regulated influx of Ca²⁺ into cells. The best characterized SOCE current is the Ca²⁺ release-activated Ca²⁺ current (I_CRAC), first described in mast cells and since recorded in several other types such as Jurkat and RBL cells. The mechanism by which depleted stores activate SOCE involves complex cellular movements of an endoplasmic reticulum Ca²⁺ sensor, STIM1, which redistributes to puncta near the plasma membrane and activates plasma membrane channels comprising ORAI1, ORAI2, and ORAI3 subunits that function as the pore-forming subunits of CRAC channels.

The importance of CRAC channels is apparent in human patients with loss-of-function or mutations in ORAI1 and STIM1 genes that abolish SOCE. These patients present with a unique condition, termed CRAC channelopathy, which is characterized by severe immunodeficiency, autoimmunity, congenital muscular hypotonia and loss of function of eccrine sweat glands. Abnormalities in CRAC function are also implicated in acute pancreatitis, abnormal cell proliferation, and breast cancer metastasis. The development of a simple, reliable and sensitive CRAC channel assay could contribute to the detection of novel therapeutic agents aimed at treating these human disorders.

In this study we have compared a variety of fluorescent calcium sensitive dyes, including the recently introduced FLIPR® Calcium 6 Assay Kit, in an endogenous CRAC channel assay in RBL cells using the FlexStation® 3 Multi-Mode Microplate Reader, a five-mode reader featuring an internal 8- or 16-channel pipettor for custom and direct transfer of reagents from a compound plate to an assay plate. We demonstrate how selection of the best dye is vital to give the most reliable, consistent, and robust assay that can be used in a high-throughput setting.

Materials and methods Cell culture

Cell culture

RBL cells were obtained from European Collection of Authenticated Cell Cultures (ECACC) and cultured in EMEM with 2 mM glutamine, 1 mM sodium pyruvate, 1% Non-Essential Amino Acids and 10% fetal bovine serum. The cells were maintained at 37°C in a humidified incubator set at 5% CO2. Cell plates were prepared by dispensing 10,000 cells per well into Corning CellBIND black wall, clear bottom 384-well microplates (cat. #3683) in 50 μL of growth media and allowed to adhere overnight in a 37°C humidified incubator set at 5% CO₂.

Dye preparation

Calcium-free loading buffer: 40 mM NaCl, 100 mM KCl, 17 mM NaHCO₃, 0.1 mM EGTA, 12 mM glucose, 1 mM MgCl₂, 5 mM HEPES buffer, pH 7.4.

FLIPR Calcium 6 Assay Kit

Remove one vial of Calcium 6 Assay Reagent (Component A) from the freezer and equilibrate to room temperature. Dissolve the contents of one Component A vial by adding 10 mL of calcium-free loading buffer. Mix by vortexing (1-2 minutes) until contents of vial are fully dissolved. It is important that contents are completely dissolved to ensure reproducibility between experiments. Add 10 mL of reconstituted Component A/buffer to 10 mL buffer for a total volume of 20 mL.

Fluo-4 NW Calcium Assay Kit

Add 10 mL of calcium-free loading buffer to one bottle of Component A. This 1X dye loading solution is sufficient for one microplate. Shake or vortex the dye loading solution vigorously for 1–2 minutes to ensure that the dye dissolves completely.

Fura-2 QBT^™ Calcium Kit

Remove one vial of Calcium 6 Assay Reagent (Component A) from the freezer and equilibrate to room temperature. Dissolve the contents of one Component A vial by adding 10 mL of calcium-free loading buffer. Mix by vortexing (1-2 minutes) until contents of vial are fully dissolved. It is important that contents are completely dissolved to ensure reproducibility between experiments. Add 10 mL of reconstituted Component A/buffer to 10 mL buffer for a total volume of 20 mL.

Dye loading

Cell plates were removed from the incubator, and growth media was removed and replaced with 30 μL of 1X dye loading solution containing 2.5 mM probenecid. Plates were incubated for two hours (Calcium 6) or one hour (Fura-2 QBT and Fluo-4 NW) at 37°C, then left at room temperature (R.T.) for 15 minutes before assaying.

Assay

Compound plates were prepared by diluting 100% DMSO stocks (inhibitor dose-response curves and controls) with calcium-free buffer containing thapsigargin to deplete endoplasmic reticulum calcium stores. The final assay concentration of thapsigargin was 1 μM. 10 μL of test compound or control (+ thapsigargin) was added, and then the plate was incubated in the dark at R.T. for 15 minutes. Compounds tested were three known inhibitors of the channel: YM-58483, La³⁺, and 2-Aminoethoxydiphenylborinate (2-APB).

To induce the Ca²⁺ signal, the FlexStation 3 reader running SoftMax® Pro 7 Data Acquisition and Analysis Software was used to add 10 μL of calcium chloride (CaCl₂, final assay concentration 1 mM), and the resultant signal was measured for two minutes with excitation wavelength 485 nm, emission wavelength 525 nm and automatic emission cut-off 515 nm for Calcium 6 and Fluo4 NW. For Fura-2 QBT, the excitation wavelengths were 340/380 nm and the emission wavelength was 510 nm, with no cut-off required.

Results

Initial experiments were done to ascertain the IC₈₀ concentration of CaCl₂ to be used in the subsequent inhibition studies. The concentration of CaCl₂ chosen was 1 mM (Figure 1).

Figure 1. Concentration-effect curves to CaCl2 in RBL cells loaded with Calcium 6 ( , EC₅₀ 0.6 ± 0.03 mM), Fluo4 NW ( , EC50 0.3 ± 0.02 mM) or Fura-2 QBT ( , EC₅₀ 0.4 ± 0.01 mM). Data are mean ± s.e.m. (n = 12) and normalized as % increase over baseline.

The data from the subsequent inhibition curves studies are shown in Figures 2, 3 and 4, and the mean IC₅₀ estimates (± s.e.m.) are displayed in Table 1 along with the average assay Z’ factors.

Figure 2. (Calcium 6): CRAC channel inhibitor assay in adherent RBL cells. Calcium 6 was loaded per protocols outlined in the methods section and inhibition curves obtained with YM-58483 ( ), 2-APB ( ) and La3⁺ ( ). Data are mean ± s.e.m. (n = 12) and normalized as % increase over baseline.

Figure 3. (Fluo-4 NW): CRAC channel inhibitor assay in adherent RBL cells. Fluo4 NW was loaded per protocols outlined in the methods section and inhibition curves obtained with YM-58483 ( ), 2-APB ( ) and La³⁺ ( ). Data are mean ± s.e.m. (n = 12) and normalized as % increase over baseline.

Figure 4. (Fura-2 QBT). CRAC channel inhibitor assay in adherent RBL cells. Fura-2 QBT was loaded per protocols outlined in the methods section and inhibition curves obtained with YM-58483 ( ), 2-APB ( ) and La³⁺ ( ). Data are mean ± s.e.m. (n = 12) and normalized as % increase over baseline.

Table 1. Mean IC₅₀ estimates obtained with YM-58483, 2-APB and La³⁺ in RBL cells loaded with different calcium sensitive dyes. Results are mean ± s.e.m (n ≥ 8).

Conclusion

We have compared three ‘no-wash’ calcium indicators in a calcium release-activated calcium (CRAC) channel assay using an RBL cell line on the FlexStation 3 reader. The Fura-2 QBT and Calcium 6 Assay Kits are homogeneous, fluorescence-based formulations designed to expedite assay development and screening of GPCR and ion channel targets, based on patented masking dye technology to reduce background fluorescence and improve the signal-to-background ratio. The Fluo-4 NW (no-wash) Calcium Assay Kit provides an alternative method for detecting intracellular calcium, but without a masking dye.

Initial experiments to determine the optimal EC₅₀ concentration of CaCl₂ showed the benefit of the FLIPR Calcium 6 Assay Kit, with a bigger and more robust fluorescence signal than the other two assay kits. The EC₅₀ values for CaCl₂ were consistent between the dyes used.

A similar picture was seen when comparing antagonist data generated on the FlexStation 3 reader. The pharmacological profile was consistent between the three treatment groups (Table 1), however the assay robustness, as determined by calculating the Z’ factor, did vary significantly. Data generated with the Fluo-4 NW kit tended to be less robust, and the assay fidelity was consequently lower. The Fura-2 QBT Calcium Kit employs the proprietary masking technology and benefits from being a ratiometric calcium indicator, however the highest signal, and associated Z’ factor, was obtained with the FLIPR Calcium 6 Assay Kit.

Download PDF