detection of intracellular Ca 2+ Liesbeth Lenaerts - Willy Verheyen - - PowerPoint PPT Presentation

Evaluation of Axxam’s new biosensor and comparison with the classical Fluo4 dye for the detection of intracellular Ca 2+ Liesbeth Lenaerts - Willy Verheyen - Sjoerd Van Lierop Janssen Research & Development C.R.E.A.Te Community of Research Excellence & Advanced Technology

Ca 2+ plays an essential role in physiology and biochemistry of the cell - Signal transduction - Neurotransmitter release - Contraction - ....... 2

AXXAM technology • Uses a GCaMP2.1 Ca 2+ biosensor • GCaMP2.1_Hek293 and GCaMP2.1_CHO stable cell lines are available. • Sensor is composed of cpEGFP, CaM-unit and M13pep. Ca 2+ binds CaM-unit and fluoresces. Can be used for detection of [Ca] i changes caused by Gq coupled GPCR’s, Ca 2+ ion-channels and others. • No need for extra loading with Ca 2+ indicator. • No need for probenecid or a mask dye • Serum reduces signal, wash-step can be required. 3

Classical Fluo4 technology • Cells are loaded with non-fluorescent Structure of Fluo4 acetoxymethyl ester Fluo4-AM. • Fluo4-AM is cleaved inside by esterases to Fluo4. • Ca 2+ binds Fluo4 and emits light after excitation at 488 nm • Fluo4 can be exported by organic anion transporters. • Loading dyes give higher baseline fluorescence • Addition of mask dyes to lower baseline fluorescence 4

Fluo4 technology versus AXXAM technology AXXAM Fluo4 GPCR GPCR Ca 2+ Ca 2+ Ion-channel Ion-channel Ca 2+ Ca 2+ Gq-subunit Ca 2+ Gq-subunit Ca 2+ Ex l = 488 nm Ex l = 488 nm Ca 2+ Fluo4 Ca 2+ GCaMP2.1 Biosensor ER ER Nucleus Nucleus Fluo4-AM loading (Probenecid) Em l = 530nm Em l = 520-540nm 5

FDSS6000 and its characteristics • Measures luminescence and fluorescence. • Kinetic read out • 96- and 384 pipettor. • Wash tool • Applications : - Ca 2+ based GPCR assays - fura2 - aequorin - Ca 2+ kits - ….. - membrane potential assays - ion-channel receptors - FRET assays - luminescence and fluorescence based assays - transport assays - mitochondrial membrane potential assays - …… 6

General protocol for detection of [Ca 2+ ] i with AXXAM and Fluo-4 technology AXXAM technology Fluo-4 technology Seed cells in 384 plates  • Seed cells in 384 plates Incubate cells for 20-24 h  • Incubate cells for 20-24 h Remove medium or wash cells  • (Remove medium or wash cells) Add 20 µl HBSS buffer with 0.1% BSA pH7.4  • Add 20 µl Fluo4 loading buffer (HBSS with 0.1% BSA, probenecid and pH7.4) Incubate cells for 20’ at RT  • Load for ± 60’ at 37 ° C or 25 ° C (Add antagonist)  • Remove loading buffer Add agonist  • Add 20 µl HBSS buffer with 0.1% BSA, Monitor signal in FDSS6000  probenecid and pH7.4 • Incubate cells for 20’ at RT • (Add antagonist) • Add agonist • Monitor signal in FDSS6000 7

Performed experiments for evaluation A. Optimization of a FDSS protocol with ionomycin B. Comparison AXXAM – Fluo4: with Ca 2+ ionophore ionomycin: in CHO and Hek cells with overexpressed GPCR’s: GPCR_1 in HEK and CHO GPCR_2 in HEK GPCR_3 in CHO and HEK with endogenous receptors: P2Y receptor in CHO muscarinic AChR in HEK C. Testing low sera and phenolred free media to avoid quench effect or wash-step . D. Testing AXXAM technology with suspension cells. 8

Evaluation A. Optimization of a FDSS protocol with ionomycin 9

Testing response of GCaMP2.1_CHO AXXAM cell line with dose response ionomycin and  cell concentrations 4000 cw for further optimization 10

Testing response of GCaMP2.1_HEK AXXAM cell line with dose response ionomycin and  cell concentrations 6000 cw for further optimization 2000 cw 4000 cw 6000 cw 10000 cw 15000 cw 20000 cw Bottom 110.7 134.5 282.6 271.1 421.7 446 Top 536.9 1113 1710 1234 2181 2253 EC50 3.58E-07 2.83E-06 9.85E-07 3.29E-07 7.01E-07 5.69E-07 Ratio (Top/Bottom) 4.9 8.3 6.1 4.6 5.2 5.1 11

Optimization FDSS protocol Determination of exposure time for GCaMP2.1_HEK AXXAM - and GCaMP2.1_CHO AXXAM cell line 6000 cw 4000 cw HEK short expo HEK normal expo HEK long expo CHO long expo CHO short expo CHO normal expo 100ms 200ms 500ms 500ms 100ms 200ms BOTTOM 1.2 1.2 1.2 Bottom 1.2 1.2 1.1 TOP 5.7 6.1 4.1 Top 3.7 6.0 7.2 EC50 3.38E-07 3.48E-07 1.63E-07 EC50 2.15E-07 3.39E-07 3.80E-07 Ratio Ratio (Top/Bottom) 4.9 5.3 3.4 (Top/Bottom) 3.2 5.1 6.4 12

Optimization FDSS protocol Determination of dispense speed for GCaMP2.1_HEK AXXAM - and GCaMP2.1_CHO AXXAM cell line HEK AXXAM CHO AXXAM 3 mean ± std (n=6) exp time 200ms 4000 cw 6000 cw disp 15 µl/s disp 20 µl/s disp 25 µl/s disp 30 µl/s disp 15 µl/s disp 20 µl/s disp 25 µl/s disp 30 µl/s 1.1 1.1 1.2 1.1 BOTTOM 1.1 1.2 1.2 1.2 BOTTOM 7.2 7.4 7.6 7.4 TOP 6.4 6.0 5.8 6.4 TOP 3.80E-07 4.58E-07 4.61E-07 4.47E-07 4.21E-07 3.68E-07 3.98E-07 4.08E-07 EC50 EC50 Ratio Ratio 6.4 6.6 6.5 6.5 5.6 4.9 4.7 5.6 (Top/Bottom) (Top/Bottom) 13

Evaluation B) Comparison AXXAM – Fluo4 : - with Ca 2+ ionophore ionomycin: in CHO and Hek cells - with overexpressed GPCR’s: - GPCR_1 in Hek and CHO - GPCR_2 in Hek - GPCR_3 in CHO and Hek transiently transfected with efficiency of ± 70-80% ( eGFP cDNA) - with endogenous receptors: - P2Y receptor in CHO - hmuscarinic AchR in HEK 14

Comparison AXXAM- and Fluo-4 technology for detection of [Ca 2+ ] i tested with dose response ionomycin on CHO cells 4000 cw CHO-K1-WT Fluo-4 CHO Axxam Bottom 1.1 1.2 Top 4.5 5.7 EC50 1.06E-07 8.86E-08 4.1 4.6 Ratio (Top/Bottom) 15

Comparison AXXAM- and Fluo-4 technology for detection of [Ca 2+ ] i tested with dose response ionomycin on HEK cells 6000 cw HEK Axxam HEK B wt Fluo4 BOTTOM 1.046 0.9075 TOP 7.827 ? LOGEC50 -5.131 EC50 7.40E-06 Ratio (Top/Bottom) 7.5 16

Comparison AXXAM- and Fluo-4 technology for detection of [Ca 2+ ] i tested with dose response agonist on GPCR_1 overexpressing HEK cells Transiently transfected 6000 cw GPCR_1 HEK AXXAM GPCR_1 HEK B Fluo4 BOTTOM 1.1 1.1 TOP 2.5 2.3 EC50 3.54E-08 1.20E-07 Ratio (Top/Bottom) 2.2 2.2 Ratio Iono 10 µM 4.0 4.1 17

Comparison AXXAM- and Fluo-4 technology for detection of [Ca 2+ ] i tested with dose response agonist on GPCR_1 overexpressing CHO cells 4000 cw Transiently transfected 4000 cw 18

Compounds tested as AGONISTS on GPCR_1 overexpressing CHO cells Axxam versus Fluo-4 technology Mean ± std (n=4) 6000 cw comp 4 EC 50 9.67E-7 comp 4 EC 50 1.34E-6 Fluo-4 AXXAM 19

Compounds tested as PAMs on GPCR_1 overexpressing CHO cells Axxam versus Fluo-4 technology AXXAM Fluo-4 AXXAM 20

Compounds tested as agonists and PAMs on GPCR_1 overexpressing CHO cells Summary permanent transfection transient transfection (PE) CHO-K1 CHO-K1 Axxam CHO cells Fluo-4 AM loading Fluo-4 AM loading Agonism PAM Agonism PAM Agonism PAM comp 1 <4 6.2 <4 5.7 <4.5 -4.8 6.1 comp 2 <4 6.6 <4 6.5 <4.5 6.4 comp 3 <4 6.8 <4 6.5 <4.5 -4.7 6.6 comp 4 5.9 6 5.7 5.5 6.8 - 21

Comparison AXXAM- and Fluo-4 technology for detection of [Ca 2+ ] i tested with dose response agonist on GPCR_2 overexpressing HEK cells 12.000 cw Transiently transfected with GPCR_2 cDNA and Gqi5 ratio : 2/3 GPCR_2 HEK AXXAM GPCR_2 HEK B Fluo4 BOTTOM 1.0 1.1 TOP 1.6 1.3 EC50 4.17E-08 1.71E-07 Ratio (Top/Bottom) 1.5 1.2 Ratio Iono 10 µM 4.2 3.9 22

Dose response agonist on GCaMP2.1_CHO AXXAM cell line overexpressing GPCR_3 and G a 16 10 µM ionomycin 20.000 cw Fluo4 stable cell line GPCR_3/Ga16 GPCR_3/Ga16 GPCR_3/Ga16 GPCR_3/Ga16 Ratio 3.2 2 / 0 2 / 0.5 2 / 2 2 / 3 EC 50 4.37E-6 BOTTOM 1.05 1.10 1.09 1.10 TOP 1.10 2.17 2.29 2.1 EC 50 7.77E-06 7.94E-06 6.92E-06 Ratio (Top/Bottom) 2 2.1 1.9 23

Dose response agonist on GCaMP2.1_HEK AXXAM cell line overexpressing GPCR_3 / G a 16 mean ± std (n=4) 24

Compounds tested as AGONISTS and PAMs on GCaMP2.1_HEK AXXAM cell line overexpressing GPCR_3 / G a 16 mean ± std (n=4) 25

Compounds tested as AGONISTS and PAMs on GCaMP2.1_HEK AXXAM cell line overexpressing GPCR_3 / Ga16 Summary permanently transfected HEK293 Axxam HEK Fluo-4AM loading results 10/03/2010 Agonism PAM Agonism PAM comp 1 < 4.52 7 6 (partial) 7.3 comp 2 < 4.52 5 4.7 5.8 comp 3 < 4.52 6.4 5.3 6.7 comp 4 < 4.52 6.6 5.7 6.8 comp 5 < 4.52 6.4 < 4.52 6.9 agonist 5.1 6.1 26

Comparison AXXAM- and Fluo-4 technology for detection of [Ca 2+ ] i tested with dose response carbachol on endogenous musc. AchR in HEK cells HEK B Fluo4 HEK B Fluo4 HEK B AXXAM HEK B AXXAM 4000 cw 8000 cw 4000 cw 8000 cw BOTTOM 1.1 1.1 1.0 1.1 TOP 2.0 2.0 3.5 2.8 EC50 2.40E-05 1.86E-05 4.14E-06 2.64E-06 Ratio (Top/Bottom) 1.9 1.9 3.4 2.6 Ratio Iono 10 µM 4.2 3.2 4.7 3.2 27

Comparison AXXAM- and Fluo-4 technology for detection of [Ca 2+ ] i tested with dose response ATP on endogenous P2Y receptors in CHO cells CHO Fluo4 CHO Fluo4 CHO AXXAM CHO AXXAM 2000 cw 4000 cw 2000 cw 4000 cw BOTTOM 1.1 1.1 1.1 1.0 TOP 6.0 4.8 5.2 5.6 EC50 6.68E-07 7.42E-07 1.29E-06 1.40E-06 Ratio (Top/Bottom) 5.5 4.5 4.8 5.4 Ratio Iono 10 µM 8.6 5.8 6.5 6.7 28

Evaluation C) Testing low sera and phenolred free media to avoid quench effect or wash-step . 29