G protein-coupled receptors (GPCRs) represent one of the most important therapeutic targets in drug discovery research. GPCRs are membrane-localized proteins that play an important role in cell signaling. When a receptor is activated by a ligand, the conformation of the receptor is modified, activating G-proteins inside the cell. An active G-protein has the potential to induce various cascades of intracellular messengers, including calcium.
Calcium-activated photoproteins are important tools for detecting receptormediated signaling events involving calcium mobilization in mammalian cells. One major advantage of photoproteins is the immediate emission of flash luminescence upon calcium binding to the coelenterazine-photoprotein complex. The background signal of aequorin measurements is close to zero, resulting in high signal-to-background ratios. Furthermore, the light emitted by the oxidation of coelenterazine does not depend on optical excitation, eliminating issues with auto-fluorescence.
There are now a number of commercial sources for photoprotein expressing cell-lines, making this technology more widely accessible. These assay methods are also described in the literature1 and in the United States Patent 6,872,538 and European Patent 1,145,002. Users interested in the patented methods may wish to consult legal counsel in evaluating these patents.
Molecular Devices FlexStation® 3 Multi-Mode Microplate Reader has integrated fluidics, permitting the realtime measurement of fluorescent and luminescent cell-based assays. Up to eight (96-well format) or sixteen (384-well format) wells can be monitored simultaneously before, during, and after compound addition. Assays are easily optimized with user-defined dispense height, speed of compound addition, reagent source location, and tip selection for each addition. Disposable pipette tips reduce the opportunity for cross-contamination between additions and experiments, and decrease plate dead volumes (compared to injectors) saving precious reagents.
The FLIPR Tetra® System from Molecular Devices is the market-leading instrument for monitoring GPCR and ion channel activity, and represents a reliable and flexible HTS/uHTS solution for early identification of lead compounds in the drug discovery process. The FLIPR Tetra System is now available with an aequorin option which includes a novel ICCD camera technology optimized for use with both fluorescent and luminescent assays. Furthermore, the cell suspension system makes the system amenable to both adherent and suspension cell-based assays in 96-, 384- and 1536-well formats.
This application note provides a basic protocol for performing an adherent aequorin assay using the FlexStation 3 reader and FLIPR Tetra System with ICCD camera. Both instruments were used to determine the concentration-response of IMETIT in CHO mito-Photina/H3 cells at various cell concentrations.
- CHO mito-Photina/H3 cells (Axxam SpA). The H3 cell line was generated by stably transfecting CHO-K1 cells with Photina, a mitochondrial membrane-targeted chimeric photoprotein, with Ga16, and with the Histamine H3 gene
- Culture media: Dulbecco’s MEM/ Nutrient Mix F12 with 15 mM HEPES (Cat. #11039- 047), supplemented with 1.35 mM sodium pyruvate (Cat. #11360-070), 10% FBS (Cat. #10082-147), 1% penicillin/streptomycin (Cat. #15070-063), 500 µg/mL Geneticin (Cat. #10131-027), and 1% L-glutamine (Cat. #25030-081), all from Invitrogen.
- PBS without calcium and magnesium (Invitrogen Cat. #14190-144)
- VERSENE 1:5000 (Invitrogen Cat. #15040-066)
- Native coelenterazine (PharmaTech Int. Cat. #55770-48-1) stock solution at 11 mM diluted in DMSO (Sigma Cat.#D5879) and 1mM glutathione (Sigma Cat.#G-6529)
- Assay Buffer: DMEM/HAM’s F12 without phenol red (Invitrogen Cat. #11039-021) with 15 mM HEPES (Invitrogen Cat. #11039-047) and 0.1% BSA (Sigma Cat. #A3294)
- Histamine H3 Agonist: IMETIT (Sigma Cat. #I-135)
- 384-well black-wall, clear-bottom microplates (Corning Cat. #3712)
- FLIPR Tetra System with ICCD camera option (Molecular Devices)
- FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices)
Assay plate preparation
To create assay plates, CHO mito-Photina/ H3 cells were harvested using VERSENE, re-suspended in culture media without selection antibiotics and plated at varying cell concentrations (625, 1250, 2500, and 5000 cells/well) in 25 µL in 384-well, blackwall, clear-bottom plates and incubated overnight at 37°C in 5% CO2.
Assay plates were removed from the incubator and culture media was removed and replaced with 25 µL assay buffer containing 5 µM native coelenterazine. The coelenterazine-loaded plates were incubated at room temperature for five hours in the dark.
Compound plate preparation
Polypropylene 384-well plates were used to prepare a concentration-response series of 2X concentrations of IMETIT, a H3- Histamine receptor agonist, in assay buffer.
Instrument setup and data analysis
After incubation with coelenterazine, assay plates were placed on the FlexStation 3 reader or FLIPR Tetra System. Protocols were prepared in SoftMax® Pro Software (FlexStation 3) or ScreenWorks® Software (FLIPR Tetra) using the parameters shown in Tables 1 and 2, respectively.
|Read Mode||Luminescence, Bottom Read|
|Run Time||90 Sec. Total|
|Assay Plate Type||384 W Corning Clear/Flatbottom|
|Compound Source||Greiner Polypropylene V Base (384-Well)|
|Initial Volume||25 µL|
|Pipette Height||20 µL|
|Rate||8 (31 µL/sec)|
|Time Point||21 sec.|
|Exposure Time||0.4 sec|
|Read Interval||1 sec|
|Dispense Volume||25 µL|
|Dispense Height||10 µL|
|Dispense Speed||50 µL/sec|
|Removal Speed||10 mm/sec|
|Expel Volume||0 µL|
Luminescence readings were taken for ~15 sec. to give a baseline reading and then the instruments’ on-board fluidic systems were used to dispense different concentrations of the specific ligand (IMETIT) whilst changes in luminescence were monitored simultaneously. Calculation of average RLU (max-min), standard deviation, EC50, Z factor at EC80, and graphing of concentration response curves were all performed using SoftMax Pro Software. A preconfigured aequorin assay protocol for the FlexStation 3 reader is available in the SoftMax Pro Software.
IMETIT-stimulated calcium flux in CHO mito-Photina/H3 transfected cells may be monitored using flash luminescence. Luminescent signal is measured by both the FlexStation 3 reader and the FLIPR Tetra System as Relative Light Units (RLU) vs. time during the assay. The average RLUs (max-min) were plotted against the IMETIT concentration to generate concentration-response curves (Figure 1, FlexStation 3 reader data; Figure 2, FLIPR Tetra System data) and the calculated EC50 value.
The IMETIT EC50, and Z-factor at EC80 are summarized in Tables 3 and 4. The EC50 values for IMETIT using the FlexStation 3 microplate reader range from 1.81 to 2.67 nM , whereas the FLIPR Tetra System with ICCD camera option reported EC50 values in the range 1.18 to 3.00 nM. Both sets of results are close to the published EC50 value of 3.43 nM. Z-factor values at EC80 are also comparable between both instruments.
|Cells/Well||EC50 IMETIT (nM)||Z factor at EC80|
|Cells/Well||EC50 IMETIT (nM)||Z factor at EC80|
The FlexStation 3 reader and the FLIPR Tetra System with aequorin option (ICCD camera) have been used to measure the Photina adherent luminescent cell assay. The preparation and treatment method of Photina cells for an adherent luminescent assay is the same when using either the FlexStation 3 reader or the FLIPR Tetra System with ICCD camera option. This, along with the correlation of the data produced, supports the use of the FlexStation 3 reader in lower-throughput Photina assays. In addition, the instrument can be used to develop assays which easily transfer to the FLIPR Tetra System for high-throughput screening purposes.
The authors thank Axxam for providing Photina cell lines for this study and Sabrina Corazza and Silvia Bovolenta of Axxam for kindly reviewing the data. Special thanks to Laurence Monnet, Maki Kato, Kristin Prasauckas, Irina Osetinsky, and Kasia Paczyna for their assistance with the FLIPR Tetra System assays.
- Boie, et al., Eur J Pharmacol 340(2-3):227-241 (1997).