Running an Aequorin Cell-Based Luminescent Assay on the FLIPRTETRA® Veena Menon1, Suresh Babu Poda1, Natalia Betancourt1, Kathy Zhao2 and Vincent Dupriez2 1 Lundbeck Biological Research USA, Inc and 2 PerkinElmer Life & Analytical Sciences Kinetic measurement of Aequorin signal on the FLIPRTETRA® 4 Kinetics of CHO-Aequorin Response - FLIPRTETRA® Reagents Pe eak RLU 10 000 cells/well, black, clear bottom assay plate 500 Ligand Dispensing 3000 Double transfected aequorin cell lines, stably expressing both mitochondriatargeted aequorin and a GPCR were used in this study: Histamine H1 AequoScreen® cell line (PerkinElmer # ES-390-A), Vasopressin V1B AequoScreen®; cell line (PerkinElmer # ES-362-A), Vasoactive Intestinal Peptide VPAC1 AequoScreen® cell line (PerkinElmer # ES-273-A). In addition, the CHOK1 (+Gα16) AequoScreen® Parental cell line (PerkinElmer # ES-000-A24 ) was also used used, using ATP as an agonist of the endogenous P2Y receptor. receptor Assay Buffer was DMEM/Ham's F12 culture medium (with 15 mM HEPES, Lglutamine, without phenol red; Invitrogen Cat n°11039) + 0.1% protease-free BSA. Coelenterazine h came from Promega (Cat n° S2011): 1 mM stock solution in methanol. Digitonin came from Sigma (Cat n°37006): 50 mM stock solution in DMSO DMSO. Kinetics of CHO-Aequorin-H1 Response - FLIPRTETRA® 2000 1000 0 0 5 10 15 ATP (-3) ATP (-4) 400 ATP (-5) ATP (-6) 300 ATP (-6.5) 200 ATP (-7) ( 7) ATP (-7.5) 100 ATP (-8) Buffer 0 Digitonin 100 µM 0 Pe eak RLU 2 10 000 cells/well, black, clear bottom assay plate 4000 20 25 time (s) 30 35 40 45 Ligand Dispensing 5 10 15 20 25 30 35 40 45 Histamine (-6) Histamine (-7) Histamine (-8) Histamine (-8.5) Histamine ((-9) 9) Histamine (-9.5) Histamine (-10) Buffer Digitonin 100 µM time (s) 5 000 0 -10 -9 -8 -7 -6 -5 -4 -3 Z' (EC100 Ago vs Buffer) 6.49 6.4 6.42 6.45 0.85 0.94 0.47 0.61 3 000 1 500 000.0 Histamine Digitonin ATP 100 µM Buffer 2 000 1 000 pEC50 = 9.19 Z' (EC80) = 0.62 AUC (0 0-60sec) 10 000 c/w 5 000 c/w 2 000 c/w 1 000 c/w pEC50 AUC (Samp ples 5 to 14) 10 000 LumiLux® 5 000 Adherent Frozen Cells/Well FLIPRTETRA® 10 000 Adherent Frozen Cells/well 10 000 c/w 5 000 c/w 2 000 c/w 1 000 c/w 15 000 Pharmacology (FLIPRTETRA® vs LumiLux®) H1 CHO (+Gα16) Aequorin Parental Cell Line 1 000 000.0 pEC50 = 9.18 Z' (EC80) = 0.61 500 000.0 0 0.0 -13 -12 -11 -10 -9 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -2 Histamine Digitonin ATP 100 µM Buffer -8 -7 -6 -5 -4 -3 Log[Ligand],M Log[ligand],M Log[ATP],M CHO-Aequorin-H1 Cell Line 10 000 c/w 10 000 c/w 5 000 c/w 2 000 c/w 1 000 c/w 1 000 500 0 -11 -500 -10 -9 -8 -7 -6 pEC50 Z' (EC100 Ago vs Buffer) 9.42 9.27 9.28 9.05 0.66 0.85 0.5 < 0 V1B Ph Pharmacology l (FLIPRTETRA® vs LumiLux L iL ®) FLIPRTETRA® 10 000 Adherent Frozen Cells/Well 4 000 LumiLux® 5 000 Suspension Frozen Cells/Well Arg-8-Vasopressin Digitonin ATP 100 µM Buffer 3 000 2 000 pEC50 = 10.40 1 000 Z' (EC80) = 0.69 Log[ligand],M LumiLux® 5 000 Suspension Frozen Cells/Well FLIPRTETRA® 10 000 Adherent Frozen Cells/Well -5 Log[Histamine],M Increasing cell numbers per well were used to assess the sensitivity of the FLIPRTETRA® in relation with the brightness of the cell lines. EC50 values were unchanged by varying the cell density. The CHO (+Gα16) Aequorin parental cell line was brighter than the H1 cell line, and yielded acceptable values even when using 1,000 cells/well. For the H1 cell line, at least 2,000 cells/well were needed to get sufficient signal intensity. 6A VPAC1 2 000 000 Arg-8-vasopressin ATP 100 µM Buffer 1 500 000 pEC50 = 10.3 1 000 000 Z' (EC80) = 0.60 500 000 0 -15 15 -14 14 -13 13 -12 12 -11 11 -10 10 -9 9 -8 8 -7 7 -6 6 -5 5 -4 4 Log[ligand],M CHO-Aequorin-Vasopressin 1B receptor cells were analyzed on the FLIPRTETRA® and on the LumiLux® Cellular Screening Platform using the adherent (FLIPR®) and suspension (LumiLux®) luminescence assay protocols EC50 values obtained on both readers and in both assay protocols. modes were equivalent. VIP Digitonin ATP 100 µM Buffer 2 500 2 000 AUC (0-60sec) 1 500 10 000 c/w 5 000 c/w 2 000 c/w 1 000 c/w CHO-Aequorin Histamine H1 receptor AequoScreen® cells were analyzed on the FLIPRTETRA® and on the LumiLux® Cellular Screening Platform using the adherent luminescence assay protocols. EC50 values obtained on both readers and in both assay modes were equivalent. AUC (samples 5 to 14) 2 000 0 -15-14-13-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 Using 3 second intervals, kinetics of the emission of light can be measured by the FLIPRTETRA®. The kinetics of the responses of 2 AequoScreen® cell lines are shown. The signal intensity was well above 150 Peak RLU (dashed line), which is an acceptable th h ld lilimit. threshold it 6B Number of cells per well - sensitivity AUC (0-60sec) compounds resulting in a large percentage of false positive or false negative hits in agonist and antagonist screens respectively. D di t d hi Dedicated, highly hl sensitive iti readers d such h as th the L LumiLux iL ® ® (PerkinElmer), the MicroBetaJet (PerkinElmer), the FDSS (Hamamatsu Photonics) and the Cybi®Lumax (Cybio) have already been in use for several years to perform drug discovery research g aequorin q assays. y Here,, we show that selected aequorin q cell using lines generating intense luminescent signal allow comfortable detection with a less sensitive, non-luminescence dedicated reader, i.e. the FLIPRTETRA® (Molecular Devices). The FLIPRTETRA® was built for fluorescent signal detection, but by putting off the excitation light, can also be used for strong luminescent signal detection detection. We show several examples of aequorin signal detection with the FLIPRTETRA®, generating high quality data, with Z’ values compatible for HTS use of Aequoscreen® assay on the FLIPRTETRA®. Adherent cells assay: For FLIPRTETRA® measurements, cells were tested mainly in adherent mode (10 000 cells/well unless otherwise indicated), but some suspension cell assays were also performed. Cells (frozen vial) were thawed and plated in TC-treated assay plates (in Ham’sF12 with 10% serum, no antibiotics) and left in the incubator overnight (37°C, 5% CO2). The next day, medium was removed by plate overthrow and tapping on a paper towel, then 20 μL/well of Assay Buffer containing 10 μM coelenterazine h was added to the cells and plates were incubated for 4h at RT° in the dark. Ligands (20μL/well), diluted in Assay B ff were dispensed Buffer, di d on the h cells ll using i the h FLIPR®. We W usedd black, bl k clear l bottom b 384384 well assay plates as we observed a background signal that was variable from well to well when using white assay plates (not shown). Suspension cells assay: For LumiLux® measurements, cells were tested in suspension mode (5 000 cells/well). Cells (frozen vial) were thawed and resuspended in 10-ml of assay buffer containing co ta g 5 μM μ coe coelenterazine e te a e h.. Thiss ce cell suspension suspe s o was put in a 10-ml 0 Falcon a co tube, fixed onto a rotating wheel and incubated for 4 h or overnight at RT° in the dark (8 rpm; 45° angle). Cells were diluted with Assay Buffer to 5 000 cells/20 µL. Ligands (20μL/well), diluted in Assay Buffer, were prepared in black, clear bottom assay plates, and the cell suspension was dispensed on the ligands using the LumiLux®. Digitonin at a final concentration of 100 µM diluted in Assay Buffer was used to measure th receptor-independent the t i d d t cellular ll l calcium l i response (cell ( ll membrane b permeabilization). bili ti ) The “Top/digitonin” response (expressed in percentage) is the ratio between the maximal response to the ligand of the receptor (Top) and the digitonin response (Digitonin), which is indicative of the aequorin content of the cells. FLIPRTETRA® settings: For using the FLIPRTETRA® in luminescence mode, the excitation light g was disabled ((select “Exc.WLength: g NONE” in the software), ), a ggain of 240 and an exposure time of 3 seconds were used. 20 µL of ligands were dispensed form the source plate 1 to the read plate containing the coelenterazine-loaded cells. 33 intervals of 3.1 seconds were read, 3 of them being read before ligand dispensing. Area under the curve (AUC) was calculated with the ScreenWorks™ software, using the third interval for background subtraction (“subtract bias based on sample 3”). AUC (Sa amples 4-33) Aequorin based Ca2+ assays Aequorin-based represent a new paradigm in drug discovery research for Ca2+-coupled GPCRs and ion channels cell-based assays. The inherent limitations of fl fluorescent t dye-based d b d screening i result in lower throughput and higher resource expenditure in comparison to the aequorin assay. Furthermore, g the fluorescence in a HTS setting assay approach is prone to interference from autofluorescent 5 AequoScreen® assay AUC (Sample es 5-14) 3 Introduction AUC (samples 5 to 1 14) 1 1 500 pEC50 = 8.67 1 000 Z' (EC80) = 0.58 500 400 000 VIP Digitonin ATP 100 µM Buffer 300 000 200 000 pEC50 = 7.90 100 000 Z' (EC80) = 0.71 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 Log[ligand],M Log[Ligand],M CHO-Aequorin Vasoactive Intestinal Peptide VPAC1 receptor AequoScreen® cells were analyzed on the FLIPRTETRA® and on the L iL ® Cellular LumiLux C ll l S Screening i Pl Platform tf using i th the adherent dh t (FLIPR®) and d ® suspension (LumiLux ) luminescence assay protocols. EC50 values obtained on both readers and in both assay modes were equivalent. 7 Conclusion We have shown here that signal intensity for the selected catalog aequorin cell lines is strong enough to allow its measurement by the non-luminescence dedicated FLIPRTETRA®. In particular, particular signal intensity of the CHO (+Gα16) aequorin parental cell line was very high, and thus will undoubtedly generate double-transfectant cell lines that will be suitable to use on the FLIPRTETRA®. Pharmacology and Z’ values observed are fully compatible with the use of these cell lines for HTS or profiling fili studies. di All trademarks and registered trademarks are the property of their respective owners. Lundbeck Research USA, Inc., 215 College Road, Paramus, NJ 07652; www.lundbeck.com - PerkinElmer LAS, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 (+1) 203 925-4602 www.perkinelmer.com