Practical use of JC-1 in a Drug Discovery setting

Per O.G. Arkhammar

BioImage A/S

Mørkhøj Bygade 28DK-2860 SøborgDenmark

Molecular Devices4th International Cell Analysis Products Users MeetingMay 30 – June 2, 2000

Energised

λ527 nm 590 nm

JC-1 J-aggregate

Cationic carbocyanine potentiomentric dyeJC-1: single excitiation, dual emission.

Cationic carbocyanine potentiomentric dyeJC-1: single excitiation, dual emission.

DepolarisedEnergised

488 nm

50 nM FCCPPre-treatment

1

1.5

2

2.5

3

3.5

4

4.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

JC-1

em

issi

on

575 - 625 nm

515 - 545 nm

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Minutes

JC-1

(R/G

) rat

io

Red:Green ratio corrects for bleach effectsand increases sensitivity to ∆Vmit

100 nMOligomycin

4 nM20 nM

52 nM

132 nM

Progressive uncoupling with FCCP

Ratio

1

1.5

2

2.5

3

3.5

4

4.5

1

1.5

2

2.5

3

3.5

4

4.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

JC-1

em

issi

on

575 - 625 nm

515 - 545 nm

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Minutes

JC-1

(R/G

) rat

io

Red:Green ratio corrects for bleach effectsand increases sensitivity to ∆Vmit

100 nMOligomycin

4 nM20 nM

52 nM

132 nM

Progressive uncoupling with FCCP

Ratio

IM spaceP-phase

Matrix(N-phase)

UQ

Cyt c

NADH/NAD +

Succ/Fum

? O2/H2O

I

IV

III

H+

H+

H+

ATP

F0F1 ATPaseH+ Oligomycin

X

IIX

X

XRotenone

Antimycin A

CN-

e-REV ERSIBLE

REV ERSIBLE

REV ERSIBLE

-280 mV

+390 mV

-30 mV

H+Uncoupler

IM spaceP-phase

Matrix(N-phase)

UQ

Cyt c

NADH/NAD +

Succ/Fum

? O2/H2O

I

IV

III

H+

H+

H+

ATP

F0F1 ATPaseH+ Oligomycin

X

IIX

X

XRotenone

Antimycin A

CN-

e-REV ERSIBLE

REV ERSIBLE

REV ERSIBLE

-280 mV

+390 mV

-30 mV

H+Uncoupler From: Nicholls & Ferguson, 1992

Mitochondrial Respiratory chain:proton pumps and leaks, electron flows

FCCP dose response in A10 cells(n=7)

0

2000

4000

6000

8000

10000

12000

14000

0.1 1 10 100

FCCP Concentration (nM)

JC-1

Gre

en F

luor

esce

nce

(+S

D)

Inhibited

Control

Mitochondrial ”sensitisation” with Oligomycin and Antimycin A

JC-1 green signal dose responsesfor some metabolic toxins

Control cells

Inhibition withOligomycinand Antimycin Afor 20 minutes

JC-1 green signal dose responsesfor some metabolic toxins

• Iodoacetic acid• Ionomycin• Oligomycin• Nigericin• Valinomycin• Sodium Azide• Rotenone• KCN• Atractyloside• Dinitrophenol

D-glucose

D-glucose

G 6 P

F 1,6 DPGA 3 P

DHA P

1,3 DP G PE P

Pyruvate

Lactate

TCA

gluco-kinase

phosphofructo-kinase

phosphoglucose-isomerase

F 6 P

aldolase

triosephosphateisomerase

pyruvate-kinase

LDH

PGKPGM

G3PDH

NADHFADH2

ATP

electrontransport

ATP

Glucosemetabolism

Iodoaceticacid

Atractyloside

Ionomycin

KCNSodium AzideValinomycinOligomycinRotenone

NigericinDinitrophenolFCCP

JC-1 green signal dose responsesfor some metabolic toxins

• Iodoacetic acid• Ionomycin• Oligomycin• Nigericin• Valinomycin• Sodium Azide• Rotenone• KCN• Atractyloside• Dinitrophenol

Dinitrophenol

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0.1 1 10 100 1000

Concentration (µM)F

luo

resc

ence

Inhib

Control

JC-1 green signal dose responsesfor some metabolic toxins

JC-1 green signal dose responsesfor some metabolic toxins

Nigericin

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0.01 0.1 1 10 100

Concentration (µM)F

luo

resc

ence

Inhib

Clean

JC-1 as counterscreen for unwanted metabolic effects

JC-1 comparison of two primary hits (n=5)

0

1000

2000

3000

4000

5000

6000

0.01 0.1 1 10 100

Concentration (µM)

JC-1

flu

ore

scen

ce (

mea

n+S

D)

Compound 1C

Compound 2C

Compound 1I

Compound 2I

JC-1 screen of LOPAC library(640 compounds)

0

5

10

15

20

25

30

35

40

45

50

-30000 -10000 10000 30000 50000 70000 90000 110000

JC-1 G-R (AUC) Value

Fre

qu

ency

6%

Energised

λ527 nm 590 nm

JC-1 J-aggregate

Cationic carbocyanine potentiomentric dyeJC-1: single excitiation, dual emission.

JC-1 screen of LOPAC library(640 compounds)

0

5

10

15

20

25

30

35

40

45

50

-30000 -10000 10000 30000 50000 70000 90000 110000

JC-1 G-R (AUC) Value

Fre

qu

ency

3%

JC-1 and AB comparison

0 80 160 240 320 400 480 560 640

Compound ID

Hits

JC-1

AB21 hits

23 hits} 4 common

Practical use of JC-1 in a Drug Discovery setting

Primary screening:• Uncoupling agents• Compounds that energize mitochondria• Compounds that ”protect” mitochondria• Apoptosis• MDR assessment

Secondary / counterscreening:• Targets / events regulated by cellular metabolism

ion channelsion pumpsintracellular calcium changessecretionglycolysis, Krebs cycle

Practical use of JC-1 in a Drug Discovery setting

Acknowledgements:

BioImage A/S:Dr. Bob TerryDr. Morten HeideDr. Kurt Scudder

Molecular Devices:Dr. Frank Hafner

Molecular Devices4th International Cell Analysis Products Users MeetingMay 30 – June 2, 2000