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Mark McPate PhD | Applications Scientist | Molecular Devices

Simon Lydford | Applications Scientist Manager | Molecular Devices

Introduction

As a laboratory’s requirements often change according to ongoing research needs, having a multi-mode microplate reader that offers multiple detection modes such as absorbance, luminescence, and fluorescence has become a necessity. Having a reader that is also easily upgradeable is important, for example offering a dispense option to facilitate running cell-based assays ensures flexibility, cost savings, and a reduction in bench space versus having to purchase several dedicated instruments.

Researchers interested in transitioning from biochemical to cell-based assays usually want to extract as much information as possible from each plate, popular examples being the kinetic detection of GPCR-mediated calcium signals, and ion channel activity. Typically, in these assays, the signal reaches maximum and decays in a matter of seconds and thus must be monitored in real time, at the same time as compound injection.

Here, we show how the SpectraMax® iD3 and iD5 Multi-Mode Microplate Readers, equipped with dual injector systems, can be used to develop information-rich, fluorescence-based assays to monitor intracellular calcium changes. In addition, the use of no-wash FLIPR® Calcium 6 and 6-QF Assay Kits enhances the assay signal window, improves assay robustness, and provides the necessary flexibility to work with both adherent and suspension cells.

Two assays were developed on the SpectraMax iD3 and iD5 readers and compared to the same assays run on the industry standard FlexStation® 3 Multi-Mode Microplate Reader. Initially an adherent cell assay was established to study muscarinic acetylcholine receptors in the 1321N1 human astrocytoma cell line with the FLIPR Calcium 6 kit. Then we turned to THP-1 cells, a human monocytic suspension cell line, and developed a simple workflow to enable the study of purinergic P2Y receptors using the FLIPR Calcium 6-QF assay.

Materials

Reagents

• 1321N1 cells (ECACC cat. #86030402)
  • Culture medium: DMEM + 2mM glutamine + 10% fetal bovine serum (FBS)
• THP 1 cells (ECACC cat. #88081201)
  • Culture medium: RPMI 1640 + 2mM Glutamine + 10% Fetal Bovine Serum (FBS)
• Hanks’ Balanced Salt Solution (HBSS), with calcium & magnesium (Sigma-Aldrich cat. #55037C)
• Carbamoylcholine chloride (Sigma-Aldrich cat. #C4382)
• Acetylcholine chloride (Sigma-Aldrich cat. #A6625)
• Atropine (Sigma-Aldrich cat. #A0132)
• Pirenzepine dihydrochloride (Sigma-Aldrich cat. # P7412)
• Adenosine 5′-triphosphate (ATP) disodium salt (Sigma-Aldrich cat. #A2383)
• Uridine 5′-triphosphate (UTP) disodium salt (Sigma cat. #U6625)
• Probenecid (AAT Bioquest cat. # 20061)
• FLIPR Calcium 6 Assay Kit (Molecular Devices cat. #R8190)
• FLIPR Calcium 6-QF Assay Kit (Molecular Devices cat. #R8192)

Instruments

• SpectraMax iD3 and iD5 Multi-Mode Microplate Readers (Molecular Devices)
  • SpectraMax Injector Cartridge (Molecular Devices cat. #0200-7029)
• FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices)
  • 8-channel pipettor head kit (Molecular Devices cat. #0200-6182)

Microplates

• CellBIND 96-well black wall, clear-bottom plates, sterile (Corning cat. #3340)
• Greiner 96-well plates, round-bottom with clear wells, non-sterile (Greiner cat. #650201)

Methods

Cell preparation

1321N1 “assay ready” frozen cells were thawed rapidly and seeded into a 96-well black-wall, clear-bottom plate at 2 x 10⁴ cells per well in 200 μL of supplemented DMEM media. They were incubated at 37°C, 95% humidity, and 5% CO₂ overnight.

THP-1 cells were cultured in suspension in supplemented RPMI media at 37°C, 95% humidity and 5% CO₂. On the day of use, the required number of cells for each assay was aliquoted into 50-mL centrifuge tubes and spun down at 1,000 rpm for five minutes.

Dye loading

For 1321N1 cells, culture medium was removed, and 200 μL of FLIPR Calcium 6 reagent (with probenecid) was added to each well. The plate was then incubated for 1 hour and 45 minutes at 37°C, 5% CO₂, followed by a 15 minute incubation at room temperature.

For THP 1 cells, following centrifugation the cells were resuspended in FLIPR Calcium 6-QF reagent (including probenecid) at 1.25 x 10⁶ cells per mL. Calcium 6-QF does not use the masking dye technology and is ideal for assaying cells that are not in a monolayer at the bottom of the well, but rather in true suspension (Figure 1). The cells were incubated in a shaking water bath for two hours at 37°C, and 180 μL of cell suspension was dispensed into each microplate well two minutes prior to assay.

Figure 1. FLIPR Calcium 6 and 6-QF Assay Kits. Assay flexibility with or without masking technology

Assay protocols

Agonist concentration-response curves were obtained on the SpectraMax iD3 or iD5 readers equipped with the SpectraMax Injector System with SmartInject™ Technology, one agonist concentration at a time. For each concentration-response curve, the assay was run from low to high concentration to minimize carryover. Injectors were primed with compound, then the dye-loaded cell plate was placed in the instrument and the assay was run using triplicate wells for each concentration. For 1321N1 cells, 50 µL of 5X concentrated compound was injected into each well using the SmartInject™ Technology, which shakes the microplate during injection to ensure reagent mixing. See settings, Figure 2.

Figure 2. Representative Acquisition Plan for 1321N1 cell calcium assays on the SpectraMax iD3 and iD5 readers. The Acquisition Plan Editor in SoftMax Pro 7.1 (and later) Software provides a graphical drag-and-drop interface for easy assay set up.

For the THP-1 cells, 180 μL of cells in FLIPR Calcium 6-QF reagent were pipetted into three wells of the microplate, then a similar protocol to the compound addition for 1321N1 cells was used, but with a smaller 20 μL addition of 10X agonist to minimize injection artefacts. The injector was then reversed to remove compound from the line and primed with the next highest concentration of compound. A new plate section was created, and the assay was run on the next set of triplicate wells (Figure 3).

Figure 3. Navigation tree in SoftMax Pro 7.1 Software showing the different plate sections for a 7-point concentration response curve to carbachol in 1321N1 cells on the SpectraMax iD3 and iD5 readers. One plate section was created for each concentration run.

This process was repeated for each compound concentration. The Acquisition Plan Editor in SoftMax Pro 7.1 Software was used to define the instrument and injector settings (Figure 2).

For antagonist studies (1321N1 cells only), cell culture media was removed and replaced with 180 μL per well FLIPR Calcium 6 assay reagent. The cells were incubated for 105 minutes at 37°C, followed by the addition of 20 μL of 10X concentrated antagonist, and a further 15-minute equilibration at room temperature. Agonists (5X concentration) were then added in triplicate using either injector 1 or injector 2, in a volume of 50 μL, at a pre-determined EC₈₀ concentration.

Similar dye-loading and antagonist incubation protocols were used on the FlexStation 3 reader, however the integrated 8-channel pipettor was used to deliver all seven concentrations of agonist and the buffer control from the compound plate simultaneously to every well in a column of the cell plate, or eight replicates of the agonist EC₈₀ concentration for the antagonist studies.

Results

Using the SpectraMax iD3 and iD5 readers, featuring the dual injector upgrade, we obtained robust agonist and antagonist concentration-response curves in both adherent and suspension cell lines. The kinetic data from adherent 1321N1 cells was consistent to that obtained on the medium-throughput FlexStation 3 reader and the high-throughput FLIPR® Penta High-Throughput Cellular Screening System, reaching a similar maximal response on all three systems (Figure 4). A similar situation was seen when using THP-1 cells in suspension (Figure 6).

Figure 4. Example kinetic traces showing a concentration response to acetylcholine in FLIPR Calcium 6 reagent loaded 1321N1 cells on the iD5 (A), the FlexStation 3 reader (B) and the FLIPR Penta system (C).

Figure 6. Example kinetic traces showing a concentration response to UTP in THP-1 cells in suspension on the SpectraMax iD5 reader (A), the FlexStation 3 reader (B) and the FLIPR Penta system (C).

With adherent 1321N1 cells, we used the FLIPR Calcium 6 reagent featuring novel masking technology to reduce extracellular background. We measured responses to the muscarinic receptor ligands acetylcholine, carbachol, atropine, and pirenzepine. The 4-parameter logistic curve fit built into SoftMax Pro Software, and a custom data reduction, were used to calculate agonist EC₅₀ values, as well as antagonist IC₅₀ values (Figure 5).

Figure 5. Representative pharmacological profile of FLIPR Calcium 6 reagent loaded 1321N1 cells endogenously expressing muscarinic receptors. Acetylcholine (●), carbachol (●), atropine (●) and pirenzepine (●)

The calculated EC₅₀ and IC₅₀ values obtained on the SpectraMax iD3 and iD5 readers are shown in Table 1, alongside the equivalent data obtained on the FlexStation 3 reader and FLIPR Penta system. There were no significant differences observed across all three instruments.

Table 1. Agonist and antagonist data obtained from Calcium 6 dye-loaded 1321N1 cells. All data are in μM and are n ≥ 3 independent experiments, with the exception of data labelled*, which is n = 2. Data from the FLIPR Penta system were generated in previous studies.

Previously, for suspension cells, we have used the traditional method of creating a monolayer of dye-loaded cells on the bottom of the microplate, and then gently adding ligands to initiate the calcium reaction without adversely affecting the cell layer. In order to save on assay preparation time and cell culture consumables, but still generate robust signals, we developed a workflow based on the use of the Calcium 6-QF option. This allowed us to keep cells in suspension without interference from the quencher, and we adjusted assay volumes to minimize disruption yet still provide a good signal window (see Figure 6). The 4-parameter curve fit was used to calculate agonist EC₅₀ values (Figure 7).

Figure 7. Representative agonist concentration-response curves in Calcium 6QF loaded THP-1 cells endogenously expressing purinergic receptors for UTP (●) and ATP (●) on the SpectraMax iD5 reader.

The calculated EC₅₀ for UTP and ATP are shown in Table 2, alongside the equivalent data obtained on the FlexStation 3 reader and FLIPR Penta system. There were no major differences observed across all three instruments.

Table 2. Agonist EC₅₀ values obtained from Calcium 6-QF dye-loaded THP-1 cells. All data are in μM and are n ≥ 3 independent experiments

Conclusion

Using the SpectraMax iD3 and iD5 readers with the SpectraMax Injector Cartridge with SmartInject Technology, and FLIPR Calcium 6 and 6-QF Assay Kits, we have demonstrated that it is possible to develop robust and reproducible kinetic assays in both adherent and suspension cell lines.

For adherent 1321N1 cells using the FLIPR Calcium 6 reagent with masking technology, both agonist and antagonist data align closely with that seen on medium- and high-throughput systems such as the FlexStation 3 reader and FLIPR Penta system. Additionally, for THP-1 cells, we have shown for the first time that it is possible to develop a novel workflow on the iD3 and iD5 readers with the quench-free (QF) formulation of the FLIPR Calcium 6 assay to enable the use of cells in suspension when developing intracellular Ca²⁺ assays.

Furthermore, the data obtained on the SpectraMax iD3 and iD5 readers correlates well with data obtained on the FlexStation 3 reader and FLIPR Penta system. The readers represent affordable and upgradeable systems that enable researchers to perform ELISA and cell viability assays out of the box. The simple addition of injectors enables development of information-rich, cell-based kinetic assays that are comparable with those developed on higher-throughput systems.

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