SHAPE Society

1. Detection of production of
Reactive Oxygen Species (ROS)
by our SPIO
Preliminary Data

2. Background
• Since the production of ROS in the plaque
would have significant effect on the
vulnerability of the plaque, we wanted to
check if our SPIO would produce ROS.

3. Principle
• Any event of phagocytosis is immediately
followed by a transient release of super oxide due
to the assembly of the NADPH oxidase against the
plasma membrane. Subsequently the oxidase
translocates onto the phagosomes containing the
SPIO to produce intracellular ROS, which is
secreted extracellularly.
• Thus an early extra cellular secretion of super
oxide is detectable (using luminol) soon after
phagocytosis and a later event of intracellular
secretion is measurable using DCFDA dye .

4. Method
∀ • The suspension of SPIO (1.25-10 uL) was added
to macrophages (1x10*4/well in 96 well plates).
Cells were incubated for 1 h and washed to
remove extra cellular SPIO. For each dose three
wells were tested.
Isoluminol substrate was added and super oxide
induced luminescence measured at 15, 30 and 45
min intervals using a luminometer.

5. Results
• SPIO was phagocytosized by macrophages
as early as 60 min after addition.
• Uptake was followed by release of super
oxide for all four doses tested.
Super oxide was released by SPIO at all
doses tested (1.25-10 ul)

6. Dosage of SPIO-1.25micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min NOSPIO
Sample1
Sample2
Sample3

7. Dosage at 2.5micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min
Sample1
Sample2
Sample3

8. Dosage at 5micL
0
500
1000
1500
2000
2500
3000
15min 30min 45min
Sample1
Sample2
Sample3

9. Dosage at 10micL
0
200
400
600
800
1000
1200
1400
1600
1800
15min 30min 45min
Sample1
Sample2
Sample3

10. Detection of intracellular ROS
with DCFDA dye
• Particles ingested by macrophages are enclosed
within a phagosome and the NADPH oxidase
assembles on these phagosomes to generate super
oxide. Super oxide dismutates into hydrogen
peroxide rapidly within the macrophage. This
H2O2 is detected by a fluorescent dye DCFDA.
DCFDA is by itself non-fluorescent. When it
enters a viable cell it is hydrolyzed by esterases
into DCF which is then oxidized by H2O2 to yield
a green fluorescence. This is measured by
fluorometery.

11. Method
∀• The suspension of SPIO (1.25-10 uL) was
added to macrophages (1x10*4/well in 96
well plates). Cells were incubated for 24 h
and washed to remove extra cellular SPIO.
For each dose three wells were tested.
∀• DCFDA substrate was added and read for
intracellular fluorescence using a
fluorometer at 485nm/538 nm.

12. Results
∀•Uptake was followed by release of H2O2
for all four doses tested (1.25-10micL).
∀•Note untreated macrophages have a
background fluorescence of =/< 2 AFU

13. Dosage at 1.25micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3

14. Dosage of 2.5micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3

15. At 5micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3

16. At 10micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3

17. Detection of macrophage
viability after ROS production
• Viable macrophages convert the Alamar
Blue (AB) dye into an orange colored
product measurable in a Elisa reader. AB is
a widely used method to measure viability.
• The hypothesis we are testing is that cells
that make ROS may die and lose viability.
Thus if SPIO stimulates ROS within the
cells does it lead to cell death over time ??

18. Method
∀ • The suspension of SPIO (1.25-10 uL) was added
to macrophages (1x10*4/well in 96 well plates).
Cells were incubated for 1 h or 24 h and washed to
remove extra cellular SPIO. For each dose three
wells were tested.
∀ • AB dye at 20% volume was added to the
macrophage plate and incubated for 4 h at 37o
C
and 5% Co2. They were read for viability using
an Elisa at 570 nm

19. Alamar Blue
• AB is an aqueous dye which is used to
detect cell viability and proliferation.
• Its is reduced by the intracellular
NADPH/FADPH and emits a pink
fluorescence, indicating the viability of the
cell.

20. Results
• Both SPIO treated as well as untreated
macrophages had same level of viability as
shown in the following slide at 24 h post
SPIO incubation.

21. Viability values with alamar blue
0.585
0.59
0.595
0.6
0.605
0.61
0.615
0.62
0.625
0.63
1.25mL 2.5mL 5mL 10mL Untreated
Sample1
Sample2
Sample3

22. Future plan
• As ox-LDL is known to produce ROS, we
would like to see the difference in the
production of ROS in the presence of our
SPIO.