Development of an Epic Assay for an Endogenous Purinergic P2Y Receptor in CHO and PC12 Cells
Development of an Epic® Assay for an Endogenous Purinergic P2Y Receptor in CHO and PC12 Cells
Authored by: Lucinda Gedge, Ph.D., Corning Incorporated Life Sciences, Fontainebleau, France
Originally published in SnAPPShots: A brief technical report from the Corning Applications Group. Reprinted with permission.
The Corning Epic System enables high-throughput, label-free detection in both cell-based and biochemical assays. The Epic System consists of an ANSI/SBS-standard 384-well microplate with integrated optical biosensors and a high-throughput screening (HTS) compatible microplate reader that can be operated as a standalone device for assay development or integrated with other HTS equipment.
Current approaches to study seven transmembrane proteins often involve monitoring the levels of second messengers, following dynamics of fluorescent proteins or by reporter gene assays . Cell signaling is an integrated process and crosstalk between pathways is a common phenomenon. The Epic System gives an insight into the integrated effects of a compound as the quantitative downstream signal results from cell morphology changes and mass redistribution of proteins in the cell. These cell morphology and protein redistributions changes are termed dynamic mass redistribution (DMR) .
The ability of the Epic System to detect endogeneous receptor signaling means that assays can be carried out with genetically unmodified cells. This obviates the need to generate stable cell lines over-expressing the receptor of interest. Stable cells lines are time-consuming to generate and not all proteins lend themselves to stable expression of certain genes due to toxicity issues. It is also recognized that engineering cells or loading cells with dyes may interfere with the correct physiology of the target protein. The Epic technology circumvents these problems as endogenous receptors can be screened. In addition, issues surrounding intellectual property (IP)/licensing can be avoided.
P2 purinoceptors in PC12 neuroblastoma cells (PC12 cells) are activated by ATP coupled to Ca2+ influx and catecholamine release, in addition to adenosine receptors coupled to adenylyl cyclase . Handling of these cells is problematic as the American Type Culture Collection (ATCC®) describe PC12 cells as forming loosely adherent aggregates in culture. In this application note we address the development of a cell-based assay for discovery of agonists to stimulate the endogenous P2Y receptor in standard Chinese hamster overy (CHO) cells and the loosely adherent neuroblastoma cell line, PC12.
Undifferentiated PC12 cells (between passage 6 and 20) were cultured in RPMI media supplemented with 10% fetal calf serum and 5% horse serum. Cells were cultured to 70% confluence and split 1:3/1:5. To avoid clumping, cells were harvested in 10 mL Accutase™ (Sigma Cat. No. A6964), incubated for 15 min at 37°C, spun at 800 rpm for 3 minutes and resuspended with 40 mixes in 1 mL media. Cells were then diluted to 10 mL with media and counted using trypan blue exclusion to assess viability.
PC12 cells were seeded into collagen-coated Epic microplates in serum containing medium at an optimized density of 30,000 cells/well (5k to 60k cells/well were investigated).
CHO cells were cultured in IMDM supplemented with 10% fetal calf serum and 100 U/mL penicillin/100 μg/mL streptomycin. Cells were harvested using standard trypsinization.
Cells were seeded into Epic® microplates (Cat. No. 5040) in serum containing medium at an optimized density of 15,000 cells/well.
Post-seeding, both cell lines were incubated at room temperature for 30 min prior to overnight incubation in 5% CO2 at 37°C. The following day, cells were observed microscopically to ensure correct morphology and the absence of contamination.
PC12 cells were washed four times using 25 μL/well in assay buffer (20 mM HEPES, HEPES buffered saline solution (HBSS) and 0.1% DMSO). Washing was carried out using a CyBi®-well pipettor (see Table 1 for liquid handling parameters). The final volume was 30 μL/well. Dispensing was carried out on the side of the well to avoid disturbing the cell layer close to the sensor.
CHO cells were washed using our standard cell-based assay protocol using a BioMek NX®. Cells were washed once using 50 μL/well. Parameters used are outlined in Table 1.
The Epic microplates were then equilibrated for at least 1.5 hours inside the Epic instrument on the carousel at 26°C. A baseline measurement was taken followed by addition of 10 μL ATP in assay buffer. Compounds were dispensed using CyBi-well for both cell lines with parameters outlined in Table 2.
A kinetic measurement was then carried out. In some cases, a second kinetic measurement was taken following re-addition of agonist at the same piston speed.
Kinetic results were observed using Epic Viewer and the difference between the last baseline measurements and the signal max was used to determine the EC50. Graphs were generated using GraphPad Prism® v5.01.
ATP Stimulation of CHO and PC12 Cells Generates Gq-like Signatures
Epic assays can be used to measure the response of cells to a compound in a kinetic mode. Once the kinetics of the signal have been established, assays can be set up with a single end point read to permit interleaving of plates for HTS campaigns. CHO and PC12 cells were stimulated with ATP and the response was measured using an Epic assay over time. Figure 1 reveals that for both cells lines, a fast peak characteristic of a Gq response was observed, consistent with stimulation of P2Y receptors .
Dose Response with ATP
The P2Y agonist ATP was used to calculate the EC50 in a PC12 and CHO background (Figure 2). The data was calculated 11 min 5 sec and 10 min 24 sec post baseline reading for CHO and PC12 cells respectively. High-quality data with very low standard deviation was generated for CHO cells (EC50 value was calculated to be 1.94 μM [R2=0.96]). ATP stimulation of PC12 cells gave EC50 values in the same order ( EC50; [ATP] 1.4 μM [R2=0.91]). EC50 values for PC12 cells stimulated with ATP have been reported as 80 μM measuring (3H) dopamine release  and 45 μM by measuring radiolabeled calcium influx . In comparison to the literature, the EC50 value for ATP was lower in the Epic assay; it must be borne in mind that literature values were based on radioactive measurements, not DMR, which reflects more the integrated cellular response.
|Parameter||Biomek NX Parameters (CHO cells)||CyBi-well Parameters (PC12 cells)|
|Aspirate height||2.1 mm (from bottom)||2 mm (from bottom)|
|Dispense height||2.1 mm (from bottom)||5 mm (from bottom)|
|Aspirate speed||10 μL/sec||0.8 μL/sec|
|Dispense speed||10 μL/sec||0.8 μL/sec|
|Dispense position||Center||x-1.5,y0 mm (relative to the center)|
|Parameter||CHO Cells||PC12 Cells|
|Dispense height||3.5 mm (from bottom)||5 mm (from bottom)|
|Dispense speed||8.2 μL/sec||5.7 μL/sec|
|Figure 1. Epic® Assay: Effect of ATP on CHO and PC12 cells over time. Addition of ATP resulted in a peak that descended over time.|
|Figure 2. Dose response of ATP in CHO and PC12 cells. Serial dilutions of ATP lead to generation of EC50 values for both cell lines.|
- The Epic® Label-Free System can be used to measure response to agonist without the need to over-express the target receptor.
- The Epic Label-Free System generates consistent EC50 values for known agonists.
- The response profiles observed in this study are consistent with profiles of many Gq-coupled receptors in other cell lines.
- The Epic Label-Free System can be used to detect Gprotein coupled receptor (GPCR) response of challenging, loosely attached cells, such as PC12.
- ↑ Milligan, G. High-content assays for ligand regulation of Gprotein-coupled receptors. Drug Discov Today 8 (13), 579-585 (2003).
- ↑ Fang, Y., et al. Resonant waveguide grating biosensor for living cell sensing. Biophys J 91 (5), 1925-1940 (2006)
- ↑ Yakushi, Y., et al. P2 purinoceptor-mediated stimulation of adenylyl cyclase in PC12 cells. Eur J Pharmaco 314 (1-2), 243-248 (1996)
- ↑ Fang, Y., G. Li, and A.M. Ferrie. Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells. J Pharmacol Toxicol Methods 55 (3), 314-322 (2007)
- ↑ Sela, D., E. Ram, and D. Atlas. ATP receptor. A putative receptor-operated channel in PC-12 cells. J Biol Chem 266 (27), 17990-17994 (1991)
- ↑ Michel, A.D., C.B. Grahames, and P.P. Humphrey. Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx. Naunyn Schmiedebergs Arch Pharmacol 354 (5), 562-571 (1996)
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