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Energised
527 nm
590 nm
Energised
Depolarised
Pre-treatment
50 nM FCCP
Red:Green ratio corrects for bleach effects 4.5 and increases sensitivity to Vmit
JC-1 emission
4 3.5 3
515 - 545 nm
2.5 2 1.5 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13
575 - 625 nm
14 15 16
1.9
Ratio
4 nM 20 nM 52 nM 100 nM Oligomycin 132 nM
10 11 12 13
14 15 16
Minutes
Matrix (N -phase) I
NADH/NAD +
-280 mV
Rotenone Antimycin A
eII III
Succ/Fum
-30 mV
UQ
H+
REV ERSIBLE
X
Cyt c
H+ IV H+
REV ERSIBLE
? O 2/H2O
+390 mV
CNOligomycin
X
ATP
12000 10000 8000 6000 4000 2000 0 0.1 1 10 100 FCCP Concentration (nM) Inhibited Control
Control cells
Iodoacetic acid Ionomycin Oligomycin Nigericin Valinomycin Sodium Azide Rotenone KCN Atractyloside Dinitrophenol
Glucose metabolism
D-glucose
glucokinase
G6P F6P
D-glucose
phosphoglucoseisomerase
phosphofructokinase
F 1,6 DP
aldolase
Iodoacetic acid
G3PDH
GA 3 P
1,3 DP G
DHA P
triosephosphate isomerase
PGK PGM
PE P
pyruvatekinase
Pyruvate
LDH
Ionomycin
Lactate
TCA
Atractyloside
ATP
electron transport
ATP
Iodoacetic acid Ionomycin Oligomycin Nigericin Valinomycin Sodium Azide Rotenone KCN Atractyloside Dinitrophenol
Dinitrophenol
4500 4000 3500 Inhib Control
Fluorescence
3000 2500 2000 1500 1000 500 0 0.1 1 10 100 1000 Concentration (M)
Nigericin
5000 4500 4000 Inhib Clean
Fluorescence
0.1
10
100
Concentration (M)
0.1
1 Concentration (M)
10
100
Frequency
30 25 20 15 10 5 0 -30000 -10000 10000 30000 50000 70000 90000 110000 JC-1 G-R (AUC) Value
6%
Energised
527 nm
590 nm
Frequency
30 25 20 15 10 5 0 -30000 -10000 10000 30000 50000 70000 90000 110000 JC-1 G-R (AUC) Value
3%
Hits
0
80
160
240
320
400
480
560
640
Compound ID
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 channels ion pumps intracellular calcium changes secretion glycolysis, Krebs cycle
Acknowledgements:
BioImage A/S: Dr. Bob Terry Dr. Morten Heide Dr. Kurt Scudder Molecular Devices: Dr. Frank Hafner