Cells and Geneblazer® P2RY11-NFAT-Bla CHO-K1 Cells

Version No.: GeneBLAzer® Validation Packet Page 1 of 5 01Sep08

Optimization of the GeneBLAzer® P2RY11 NFAT-bla CHO-K1 Cell Line

GeneBLAzer® P2RY11 CHO-K1 DA Assay Kit

GeneBLAzer® P2RY11 NFAT-bla CHO-K1 Cells

Catalog Numbers – K1353 and K1729

Cell Line Descriptions

GeneBLAzer® P2RY11 CHO-K1 DA (Division Arrested) cells and GeneBLAzer® P2RY11-NFAT-bla CHO-K1 cells contain the human purinergic receptor P2, G protein-coupled, 11 (P2RY11) receptor (Accession # NM_002566) stably integrated into the CellSensor® NFAT-bla CHO-K1 cell line. CellSensor® NFAT-bla CHO-K1 cells (Cat. no. K1534) contain a beta-lactamase (bla) reporter gene under control of the Nuclear Factor of Activated T-cells (NFAT) response element. Division Arrested (DA) cells are available in as an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate.

DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both GeneBLAzer® P2RY11 CHO-K1 DA cells and GeneBLAzer® P2RY11-NFAT-bla CHO-K1 cells are functionally validated for Z’-factor and ® EC50 concentrations of adenosine-5’-triphosphate (ATP); (Figure 1). In addition, GeneBLAzer P2RY11-NFAT-bla CHO-K1 cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Additional testing data using alternate stimuli are also included.

Target Description

The P2Y receptor family is part of a larger receptor family whose physiological effects are mediated by extracellular nucleotide di- and tri-phosphates. The P2 receptor family consists of ion-gated channel receptors (P2X) and G protein-coupled receptors (P2Y). Currently there are five classified P2RY receptors (P2RY1, P2RY2, P2RY4, P2RY6, and P2RY11) with additional orphan receptors, P2RY5, P2RY9, and P2RY10 (1).

The P2RY11 receptor was originally cloned from human placenta and has 33% amino acid homology to P2RY1 and 28% amino acid homology to P2RY2 (2). P2RY11 expression is broadly distributed and has been found in the brain, pituitary, lymphocytes, spleen, intestines, macrophages, lung, stomach, adipose, pancreas, kidney, prostrate, heart, placenta, liver, skeletal muscle (3). Cell differentiation of human promyelocytic HL60 cells (4) and maturation of monocyte-derived dendritic cells have been shown to be mediated by ATP activation of P2RY11 (5).

The P2RY11 receptor signaling is coupled to both the phospholipase C and cAMP pathways (2, 6, 7). The primary endogenous agonist for P2RY11 is ATP with minimal activity in response to ADP, while uridine nucleotides were initially thought to be inactive. UTP has recently been shown to be linked to an increase in cytosolic Ca2+ concentration via a mechanism independent of the signaling pathways activated by ATP (9).

Have a question? Contact our Technical Support Team NA: 800-955-6288 or INTL: 760-603-7200 Select option 3, ext. 40266 Email: [email protected] Optimization of the GeneBLAzer® P2RY11 NFAT-bla CHO-K1 Cell Line Page 2 of 5

Validation Summary Primary Agonist Dose Response

® Testing and validation of this assay was evaluated in Figure 1 — GeneBLAzer P2RY11 CHO-K1 DA and GeneBLAzer® P2RY11-NFAT-bla CHO-K1 dose response a 384-well format using LiveBLAzer™-FRET B/G to ATP under optimized conditions Substrate. 110 100 Dividing Cells 1. ATP agonist dose response under 90 DA Cells optimized conditions 80 70 60 DA cells Dividing Cells 50 EC 1 µM 1.2 µM 40 50 30

Z’-factor 0.88 0.74 % Activation 20 10 Recommended cell no. = 10K cells/well 0 -10 DMSO Tolerance = up to 1.0% -8 -7 -6 -5 -4 Recommended Stim. Time = 5 hours Log [ATP] M Max. [Stimulation] = 20 µM

® ® 2. Alternate agonist dose response GeneBLAzer P2RY11 CHO-K1 DA cells and GeneBLAzer P2RY11-NFAT-bla CHO-K1 cells (10,000 cells/well) were plated in a 384-well format and incubated for 16-20 hours. Cells UTP EC50 = N/A were stimulated with a dilution series of ATP in the presence of ADP EC50 = 7.6 µM 0.5% DMSO for 5 hours. Cells were then loaded with UDP EC50 = N/A LiveBLAzer™-FRET B/G Substrate for 2 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a 3. Antagonist dose response standard fluorescence plate reader and % Activation plotted for each replicate against the concentrations of ATP(n=6 for each data point). Suramin IC50 = 7.8 µM Alternate Agonist Dose Response nd 4. Agonist 2 messenger dose response ® ATP EC50 = 403 nM Figure 2 — GeneBLAzer P2RY11-NFAT-bla CHO-K1 dose UTP EC50 = 393 nM response to ATP, UTP, ADP and UDP under optimized ADP EC50 = 3.2 µM conditions UDP EC = 17 µM 50 120 110 UTP 100 ADP 90 80 UDP 70 ATP 60 Assay Testing Summary 50 40 30 % Activation 20 10 0 5. Assay performance with variable cell -10 -9 -8 -7 -6 -5 -4 -3 number Log [Agonist] M

GeneBLAzer® P2RY11-NFAT-bla CHO-K1 cells were plated at 6. Assay performance with variable 10,000 cells/well in a 384-well plate and incubated for 16-20 hours. Cells were stimulated with a dilution series of stimulation time adenosine-5’-triphosphate (Sigma cat# A7699), uridine-5’- triphosphate (Sigma #U1006), adenosine-diphosphate (Sigma #A2754), and uridine-diphosphate (Sigma #U4125) in the 7. Assay performance with variable presence of 0.5% DMSO for 5 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 hours. substrate loading time Fluorescence emission values at 460 nm and 530 nm were obtained and the % Activation plotted for each cell number against the indicated concentrations of the agonists (n=8 for 8. Assay performance with variable DMSO each data point). concentration

Antagonist Dose Response Agonist 2nd Messenger Dose Response Figure 4 – GeneBLAzer® P2RY11-NFAT-bla CHO-K1 dose Figure 3 — GeneBLAzer® P2RY11-NFAT-bla CHO-K1 dose response to ATP, UTP, ADP, and UDP as determined by response to Suramin measurement of intracellular Ca2+ using Fluo4

110 110 100 100 UTP 90 90 ADP 80 80 UDP 70 70 60 60 ATP 50 50 40 40 30 30 % Inhibition 20 % Activation 20 10 10 0 0 -10 -10 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [Suramin] M Log [Agonist] M

GeneBLAzer® P2RY11-NFAT-bla CHO-K1 cells were plated 16-20 GeneBLAzer® P2RY11-NFAT-bla CHO-K1 cells (10,000 hours prior to assay at 10,000 cells per well in a black-walled, cells/well) were plated in a black-walled, clear bottom 384-well clear bottom 384-well plate. A dilution series of Suramin (Sigma plate and incubated for 16-20 hours. Cells were loaded with #S2671) in the presence of 0.5% DMSO was then added to the Fluo4-AM and incubated at 37°C for 60 minutes followed by a 30 minutes at room temperature. Cells were stimulated with a cells. The cells were incubated at 37ºC with 5% CO2 for 30 min. dilution series of adenosine-5’-triphosphate (Sigma #A7699), ATP (Sigma #A7699) was added to the plate at the EC 80 uridine-5’-triphosphate (Sigma #U1006), adenosine- concentration of 3.0 µM. Cells were incubated for 4.5 hours and diphosphate (Sigma #A2754), and uridine-diphosphate (Sigma loaded for 2 hours with LiveBLAzer™-FRET B/G Substrate. #U4125) with the relative fluorescence determined by the Fluorescence emission values at 460 nm and 530 nm were FDSS every second for 180 seconds. The maximum minus obtained using a standard fluorescence plate reader and the % minimum relative fluorescent values were obtained and the % Inhibition shown plotted against the concentrations of the Activation plotted for each concentration of agonist (n=8 for antagonists. (n=16 for each data point). each data point).

Assay Performance with Variable Cell Number Assay Performance with Variable Substrate Loading Times Figure 5 – GeneBLAzer® P2RY11-NFAT-bla CHO-K1 dose response to ATP with 2.5, 5, 10, and 20K cells/well Figure 7 – GeneBLAzer® P2RY11-NFAT-bla CHO-K1 dose response to ATP with 1, 1.5, 2, and 2.5 hour substrate 45 20K cells/well loading times. 40 10K cells/well 35 20 5K cells/well 60 min 30 2.5K cells/well 90 min 25 15 20 120 min 15 150 min 10

Response Ratio Response 10 5 5

0 Ratio Response -9 -8 -7 -6 -5 -4 -3 log [ATP] M 0 -8 -7 -6 -5 -4 ® GeneBLAzer P2RY11-NFAT-bla CHO-K1 cells were plated at log [ATP] M 2500, 5000, 10000, or 20,000 cells/well in a black-walled, clear bottom 384-well plate and incubated for 16-20 hours. GeneBLAzer® P2RY11-NFAT-bla CHO-K1 cells (10,000 Cells were then stimulated with a dilution series of ATP (Sigma cells/well) were plated in a black-walled, clear bottom 384-well #A7699) in the presence of 0.1% DMSO for 5 hours. Cells plate and incubated for 16-20 hours. Cells were then were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 stimulated with a dilution series of ATP (Sigma cat#7699) in hours. Fluorescence emission values at 460 nm and 530 nm the presence of 0.5% DMSO for 5 hours. Cells were then were obtained and the Response Ratios plotted for each cell loaded for either 1, 1.5, 2, or 2.5 hours with LiveBLAzer™- number against the indicated concentrations of ATP (n=8 for FRET B/G Substrate. Fluorescence emission values at 460 nm each data point). and 530 nm were obtained using a standard florescence plate reader and the Response Ratios plotted for each substrate loading time against the indicated concentrations of ATP (n=8 for each data point).

Assay Performance with Variable Stimulation Assay Performance with Variable DMSO Time Concentration

® Figure 6 – GeneBLAzer® 2RY11-NFAT-bla CHO-K1 dose Figure 8 – GeneBLAzer P2RY11-NFAT-bla CHO-K1 dose response to ATP with 2, 3, 4 and 5 hr stimulation times response to ATP with 0, 0.1, 0.5 and 1% DMSO

25 25 1.0% DMSO 5 hr 0.5% DMSO 20 4 hr. 20 0.1% DMSO 3 hr 15 0.0% DMSO 15 2 hr

10 10

Response RatioResponse 5 Response Ratio Response 5

0 0 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 -3 log [ATP] M log [ATP] M ® GeneBLAzer® P2RY11-NFAT-bla CHO-K1 cells (10,000 GeneBLAzer P2RY11-NFAT-bla CHO-K1 cells (10,000 cells/well) were plated in a black-walled, clear bottom 384-well cells/well) were plated in a black-walled, clear bottom 384-well plate and incubated for 16-20 hours. Cells were then plate and incubated for 16-20 hours. Cells were then stimulated with a dilution series of ATP (Sigma cat# A7699) for stimulated with a dilution series of ATP (Sigma #7699) for 5 2, 3, 4, or 5 hrs in the presence of 0.5% DMSO. Cells were hours. DMSO was added to the cells at concentrations from then loaded for 2 hours with LiveBLAzer™-FRET B/G Substrate. 0% to 1%. Cells were then loaded for 2 hours with Fluorescence emission values at 460 nm and 530 nm were LiveBLAzer™-FRET B/G Substrate. Fluorescence emission obtained using a standard florescence plate reader and the values at 460 nm and 530 nm were obtained using a standard Response Ratios plotted for each stimulation time against the florescence plate reader and the Response Ratios plotted for indicated concentrations of ATP (n=8 for each data point). each DMSO concentration against the indicated concentrations of ATP (n=8 for each data point).

References

1. Ralevic, V. and Burnstock, G. (1998) Receptors for Purines and Pyrimidines. Pharmacological Reviews, 50(3), 413-492. 2. Cummuni, D., et al. (1997) Cloning of a human purinergic PRY receptor coupled to phospholipase C and adenylyl cyclase. Journal of Biological Chemistry, 272(51), 31969-31973. 3. Moore, DJ., et al. (2001) Expression pattern of human P2Y receptor subtypes: a quantitative reverse transcription-polymerase chain reaction study. Biochim. Biophys. Acta., 1521, 107- 119. 4. Communi, D., et al. (2000) Rapid up-regulation of P2Y messangers during granulocytic differentiation of HL-60 cells. FEBS Letters, 475, 39-42. 5. Wilkin, F., et al. (2001) The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells. Journal of Immunology, 166, 7172-7177. 6. Communi, D., et al. (1999) Pharmacological characterization of the human P2Y11 receptor. British Journal of Pharmacology, 128, 1199-1206. 7. Qi, AD., et al. (2001) Differential coupling of the human P2Y11 receptor to phospholipase C and adenylyl cyclase. British Journal of Pharmacology, 132, 318-326. 8. White, PJ., et al. (2003) Characterization of a Ca2+ response to both UTP and ATP at human P2Y11 receptors: evidence for agonist-specific signaling. Molecular Pharmacology, 63, 1356- 1363.

Have a question? Contact our Technical Support Team NA: 800-955-6288 or INTL: 760-603-7200 Select option 3, ext. 40266 Email: [email protected]