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 Ca2+

Liesbeth Lenaerts - Willy Verheyen - Sjoerd Van Lierop

Janssen Research & Development

C.R.E.A.Te Community of Research Excellence & Advanced Technology

Ca2+ plays an essential role in physiology and biochemistry of the cell

2

• Signal transduction
• Neurotransmitter release
• Contraction
• .......

AXXAM technology

• Uses a GCaMP2.1 Ca2+ biosensor
• GCaMP2.1_Hek293 and GCaMP2.1_CHO

stable cell lines are available.

• Sensor is composed of cpEGFP, CaM-unit

and M13pep. Ca2+ binds CaM-unit and fluoresces. Can be used for detection of [Ca]i changes caused by Gq coupled GPCR’s, Ca2+ ion-channels and others.

• No need for extra loading with Ca2+

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

acetoxymethyl ester Fluo4-AM.

• Fluo4-AM is cleaved inside by esterases

to Fluo4.

• Ca2+ 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

Structure of Fluo4

Fluo4 technology versus AXXAM technology

5

Ex l =488 nm

Gq-subunit

Nucleus ER

Ca2+ Ca2+

Ion-channel

Ca2+

GPCR Ca2+ Fluo4 Em l = 520-540nm Fluo4-AM loading (Probenecid)

Gq-subunit

Nucleus

ER

Ca2+ Ca2+

Ion-channel

Ca2+

GPCR Ca2+ Em l = 530nm GCaMP2.1 Biosensor

Fluo4 AXXAM

Ex l =488 nm

FDSS6000 and its characteristics

• Measures luminescence and fluorescence.
• Kinetic read out
• 96- and 384 pipettor.
• Wash tool
• Applications :
• Ca2+ based GPCR assays
• fura2
• aequorin
• Ca2+ 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 [Ca2+]i with AXXAM and Fluo-4 technology

• Seed cells in 384 plates
• Incubate cells for 20-24 h
• Remove medium or wash cells
• Add 20 µl HBSS buffer with 0.1% BSA pH7.4
• Incubate cells for 20’ at RT
• (Add antagonist)
• Add agonist
• Monitor signal in FDSS6000
• Seed cells in 384 plates
• Incubate cells for 20-24 h
• (Remove medium or wash cells)
• Add 20 µl Fluo4 loading buffer (HBSS with

0.1% BSA, probenecid and pH7.4)

• Load for ± 60’ at 37°C or 25°C
• Remove loading buffer
• Add 20 µl HBSS buffer with 0.1% BSA,

probenecid and pH7.4

• Incubate cells for 20’ at RT
• (Add antagonist)
• Add agonist
• Monitor signal in FDSS6000

7

AXXAM technology Fluo-4 technology

Performed experiments for evaluation

A. Optimization of a FDSS protocol with ionomycin B. Comparison AXXAM – Fluo4:

with Ca2+ 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

10

4000 cw for further

• ptimization

Testing response of GCaMP2.1_HEK AXXAM cell line with dose response ionomycin and  cell concentrations

11

6000 cw for further

• ptimization

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

Optimization FDSS protocol

Determination of exposure time for GCaMP2.1_HEK AXXAM - and GCaMP2.1_CHO AXXAM cell line

12

HEK short expo 100ms HEK normal expo 200ms HEK long expo 500ms BOTTOM 1.2 1.2 1.2 TOP 5.7 6.1 4.1 EC50 3.38E-07 3.48E-07 1.63E-07 Ratio (Top/Bottom) 4.9 5.3 3.4 6000 cw CHO long expo 500ms CHO short expo 100ms CHO normal expo 200ms Bottom 1.2 1.2 1.1 Top 3.7 6.0 7.2 EC50 2.15E-07 3.39E-07 3.80E-07 Ratio (Top/Bottom) 3.2 5.1 6.4 4000 cw

Optimization FDSS protocol

Determination of dispense speed for GCaMP2.1_HEK AXXAM - and GCaMP2.1_CHO AXXAM cell line

13

disp 15 µl/s disp 20 µl/s disp 25 µl/s disp 30 µl/s BOTTOM

1.1 1.2 1.2 1.2

TOP

6.4 6.0 5.8 6.4

EC50

4.21E-07 3.68E-07 3.98E-07 4.08E-07

Ratio (Top/Bottom)

5.6 4.9 4.7 5.6 mean ± std (n=6) exp time 200ms 6000 cw

disp 15 µl/s disp 20 µl/s disp 25 µl/s disp 30 µl/s BOTTOM

1.1 1.1 1.2 1.1

TOP

7.2 7.4 7.6 7.4

EC50

3.80E-07 4.58E-07 4.61E-07 4.47E-07

Ratio (Top/Bottom)

6.4 6.6 6.5 6.5 4000 cw 3

HEK AXXAM CHO AXXAM

Evaluation

B) Comparison AXXAM – Fluo4 :

• with Ca2+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 [Ca2+]i tested with dose response ionomycin on CHO cells

15

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

Ratio (Top/Bottom) 4.1 4.6

Comparison AXXAM- and Fluo-4 technology for detection of [Ca2+]i tested with dose response ionomycin on HEK cells

16

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 6000 cw

Comparison AXXAM- and Fluo-4 technology for detection of [Ca2+]i tested with dose response agonist on GPCR_1 overexpressing HEK cells

17

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

Transiently transfected 6000 cw

Comparison AXXAM- and Fluo-4 technology for detection of [Ca2+]i tested with dose response agonist on GPCR_1 overexpressing CHO cells

18

4000 cw Transiently transfected 4000 cw

Compounds tested as AGONISTS on GPCR_1 overexpressing CHO cells Axxam versus Fluo-4 technology

19

Fluo-4 AXXAM

comp 4 EC50 1.34E-6 comp 4 EC50 9.67E-7

Mean ± std (n=4) 6000 cw

20

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

AXXAM AXXAM Fluo-4

Compounds tested as agonists and PAMs on GPCR_1

• verexpressing CHO cells

Summary

21

transient transfection permanent transfection (PE)

Axxam CHO cells

CHO-K1 Fluo-4 AM loading CHO-K1 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

Comparison AXXAM- and Fluo-4 technology for detection of [Ca2+]i tested with dose response agonist on GPCR_2 overexpressing HEK cells

22

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 12.000 cw Transiently transfected with GPCR_2 cDNA and Gqi5 ratio : 2/3

Dose response agonist on GCaMP2.1_CHO AXXAM cell line overexpressing GPCR_3 and Ga16

23

GPCR_3/Ga16 2 / 0 GPCR_3/Ga16 2 / 0.5 GPCR_3/Ga16 2 / 2 GPCR_3/Ga16 2 / 3 BOTTOM 1.05 1.10 1.09 1.10 TOP 1.10 2.17 2.29 2.1 EC50 7.77E-06 7.94E-06 6.92E-06 Ratio (Top/Bottom) 2 2.1 1.9

20.000 cw

10 µM ionomycin

Ratio 3.2 EC50 4.37E-6

Fluo4 stable cell line

Dose response agonist on GCaMP2.1_HEK AXXAM cell line overexpressing GPCR_3 / Ga16

24

mean ± std (n=4)

Compounds tested as AGONISTS and PAMs

• n GCaMP2.1_HEK AXXAM cell line overexpressing GPCR_3 / Ga16

25

mean ± std (n=4)

Compounds tested as AGONISTS and PAMs

• n GCaMP2.1_HEK AXXAM cell line overexpressing GPCR_3 / Ga16

Summary

26

Axxam HEK

permanently transfected HEK293 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

Comparison AXXAM- and Fluo-4 technology for detection of [Ca2+]i tested with dose response carbachol on endogenous musc. AchR in HEK cells

27

HEK B Fluo4 4000 cw HEK B Fluo4 8000 cw HEK B AXXAM 4000 cw HEK B AXXAM 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

Comparison AXXAM- and Fluo-4 technology for detection of [Ca2+ ]i tested with dose response ATP on endogenous P2Y receptors in CHO cells

28

CHO Fluo4 2000 cw CHO Fluo4 4000 cw CHO AXXAM 2000 cw CHO AXXAM 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

Evaluation

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

29

Effect of wash step on signal tested with dose response ionomycin

30

w/o wash step with wash step BOTTOM 661.4 365.8 TOP 1658 2338 EC50 4.64E-07 2.90E-07 Ratio (Top/Bottom) 2.51 6.39 w/o wash step with wash step BOTTOM 258 271.1 TOP 608.2 1234 EC50 2.82E-07 3.29E-07 Ratio (Top/Bottom) 2.36 4.55

GCaMP2.1_CHO cell line 4000 cw GCaMP2.1_HEK cell line 10000 cw

Testing  phenolred-free OptiMEM media to avoid wash step performed with dose response carbachol on GCaMP2.1-HEK cells

31

with wash step HBSS w/o wash step OptiMEM 0%FCS w/o wash step OptiMEM 0.1%FCS w/o wash step OptiMEM 1%FCS w/o wash step OptiMEM 10%FCS BOTTOM

1.1 1.0 1.1 1.1 1.1

TOP

3.7 1.7 1.8 1.8 2.2

EC50

9.01E-06 8.42E-06 1.42E-05 9.66E-06 6.07E-06

Ratio (Top/Bottom)

3.4 1.6 1.7 1.7 2.0

Ratio Iono 10 µM

4.7 2.6 2.8 2.8 2.9

10.000 cw

Testing  phenolred-free DMEM media to avoid wash step performed with dose response carbachol on GCaMP2.1-HEK cells

32

with wash step HBSS w/o wash step DMEM 0.1%FCS w/o wash step DMEM 0.1%FCS + ITS w/o wash step DMEM 1%FCS w/o wash step DMEM 10%FCS BOTTOM

1.1 1.1 1.1 1.1 1.1

TOP

3.7 1.8 1.9 1.9 2.4

EC50

9.01E-06 1.75E-05 1.79E-05 1.19E-05 1.05E-05

Ratio (Top/Bottom)

3.4 1.6 1.7 1.7 1.9

Ratio Iono 10 µM

4.7 2.1 2.3 2.3 2.6 mean ± std (n=3) 10.000 cw

Testing  phenolred-free OptiMEM media to avoid wash step performed with dose response ATP on GCaMP2.1-CHO cells

33

3000 CW 6000 CW 9000 CW 12000 CW with wash step HBSS

4.6 4.8 4.4 4.2

w/o wash step OptiMEM 0%FCS

1.1 1.2 1.1 1.2

w/o wash step OptiMEM 1%FCS

1.8 2.0 1.7 1.6

w/o wash step OptiMEM 10%FCS

2.5 2.7 2.8 2.9

3000 CW 6000 CW 9000 CW 12000 CW with wash step HBSS

• 5.8
• 5.8
• 5.8
• 5.7

w/o wash step OptiMEM 0%FCS

• 6.6
• 6.4
• 6.3
• 6.0

w/o wash step OptiMEM 1%FCS

• 6.3
• 6.3
• 6.3
• 6.2

w/o wash step OptiMEM 10%FCS

• 6.5
• 6.6
• 6.5
• 6.5

Ratio: Top/Bottom pEC50

Testing  phenolred-free DMEM/F12 media to avoid wash step performed with dose response ATP on GCaMP2.1-CHO cells

34

3000 CW 6000 CW 9000 CW 12000 CW with wash step HBSS 4.6 4.8 4.4 4.2 w/o wash step DMEM/F12 0.1 % FCS + ITS 1.8 2.5 2.8 2.7 w/o wash step DMEM/F12 1 % FCS 1.8 2.6 2.8 2.8 w/o wash step DMEM/F12 10 % FCS 2.7 2.9 2.6 2.3 Ratio: Top/Bottom pEC50 3000 CW 6000 CW 9000 CW 12000 CW with wash step HBSS

• 5.8
• 5.8
• 5.8
• 5.7

w/o wash step DMEM/F12 0.1 % FCS + ITS

• 5.3
• 5.8
• 5.9
• 6.0

w/o wash step DMEM/F12 1 % FCS

• 5.6
• 5.9
• 6.1
• 6.1

w/o wash step DMEM/F12 10 % FCS

• 6.1
• 6.4
• 6.5
• 6.4

Evaluation

D) Testing AXXAM technology with suspension cells.

35

Dose response ATP on EDTA treated GCaMP2.1-CHO AXXAM cells tested in suspension mode

36

5000 cw 10000 cw 15000 cw 20000 cw 25000 cw 30000 cw 35000 cw 40000 cw BOTTOM 3167 5492 8855 11291 8533 10401 15373 12340 TOP 12716 25096 40715 54947 38613 48073 58215 56261 EC50 1.89E-06 1.71E-06 1.76E-06 1.35E-06 1.62E-06 1.48E-06 1.22E-06 1.39E-06 Ratio (Top/Bottom) 4.0 4.6 4.6 4.9 4.5 4.6 3.8 4.6

Adherent cells Ratio ± 6.5 pEC50 ±1.5E-06

Profiles of fluorescence-read-out curves of GCaMP2.1-CHO AXXAM cell line in suspension mode after stimulation with ATP

37

5000 cw 10000 cw 15000 cw 20000 cw

Ionomycin

Profiles of normalized curves (ratio) of GCaMP2.1-CHO AXXAM cell line in suspension mode after stimulation with ATP

38

5000 cw 10000 cw 15000 cw 20000 cw

Ionomycin

Dose response carbachol on EDTA treated GCaMP2.1-HEK AXXAM cells tested in suspension mode

39

5000 cw 10000 cw 15000 cw 20000 cw 25000 cw 30000 cw 35000 cw 40000 cw Bottom

5932 8757 15210 17516 12297 13930 15279 17511

Top

13710 24633 39079 48096 31422 38168 43621 48130

EC50

6.52E-06 8.63E-06 6.45E-06 7.33E-06 6.75E-06 8.94E-06 9.28E-06 7.65E-06

Ratio (Top/Bottom)

2.3 2.8 2.6 2.7 2.6 2.7 2.9 2.7

Adherent cells Ratio ± 4-4.5 pEC50 ± 2-4E-06

Testing stability and response of EDTA treated GCaMP2.1-HEK AXXAM cells in suspension mode

40

after 30 ' after 60' after 90' BOTTOM 8757 8701 9315 TOP 24633 24915 25069 EC50 8.63E-06 7.07E-06 6.79E-06 Ratio (Top/Bottom) 2.8 2.9 2.7

General Conclusions AXXAM technology

• AXXAM technology can compete with Fluo4 technology.

Higher ratio’s are perhaps desirable >>>>> create stable cell lines

• Both Axxam cell lines gave good signal for at least 24 passages (= ±12 weeks)
• Unfortunately no stable-overexpressing-AXXAM cell line was available in-house.
• Advantages :
• no need for loading step
• can be used for suspension cells in 384-format
• no need for probenecid or quench dye addition which can affect effect or potency.
• no need for negative control correction
• extremely interesting for suspension cells in 1536-format
• no extra costs >>> Fluo4 dye or Ca2+ kits are expensive
• no need for expensive coated plates in case of suspension cells
• Disadvantages:
• serum interferes >>> reduces signal >>> wash step can be required.
• Remarks :
• the expected higher z’ value allows not to remove seeding medium
• in all probability lower sera medium can be used for stable cell lines
• preferably use suspension cells (one-day assay ! )
• more options for multiplexing.

41