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Detection of production of
Reactive Oxygen Species (ROS)
by our SPIO
Preliminary Data
Background
• Since the production of ROS in the plaque
would have significant effect on the
vulnerability of the plaque, w...
Principle
• Any event of phagocytosis is immediately
followed by a transient release of super oxide due
to the assembly of...
Method
∀ • The suspension of SPIO (1.25-10 uL) was added
to macrophages (1x10*4/well in 96 well plates).
Cells were incuba...
Results
• SPIO was phagocytosized by macrophages
as early as 60 min after addition.
• Uptake was followed by release of su...
Dosage of SPIO-1.25micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min NOSPIO
Sample1
Sample2
Sample3
Dosage at 2.5micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min
Sample1
Sample2
Sample3
Dosage at 5micL
0
500
1000
1500
2000
2500
3000
15min 30min 45min
Sample1
Sample2
Sample3
Dosage at 10micL
0
200
400
600
800
1000
1200
1400
1600
1800
15min 30min 45min
Sample1
Sample2
Sample3
Detection of intracellular ROS
with DCFDA dye
• Particles ingested by macrophages are enclosed
within a phagosome and the ...
Method
∀• The suspension of SPIO (1.25-10 uL) was
added to macrophages (1x10*4/well in 96
well plates). Cells were incubat...
Results
∀•Uptake was followed by release of H2O2
for all four doses tested (1.25-10micL).
∀•Note untreated macrophages hav...
Dosage at 1.25micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3
Dosage of 2.5micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3
At 5micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3
At 10micL
0
5
10
15
20
25
30
30min 180min
Sample1
Sample2
Sample3
Detection of macrophage
viability after ROS production
• Viable macrophages convert the Alamar
Blue (AB) dye into an orang...
Method
∀ • The suspension of SPIO (1.25-10 uL) was added
to macrophages (1x10*4/well in 96 well plates).
Cells were incuba...
Alamar Blue
• AB is an aqueous dye which is used to
detect cell viability and proliferation.
• Its is reduced by the intra...
Results
• Both SPIO treated as well as untreated
macrophages had same level of viability as
shown in the following slide a...
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
Sa...
Future plan
• As ox-LDL is known to produce ROS, we
would like to see the difference in the
production of ROS in the prese...
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Detection of production preliminary

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Detection of production preliminary

  1. 1. Detection of production of Reactive Oxygen Species (ROS) by our SPIO Preliminary Data
  2. 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. 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. 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. 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. 6. Dosage of SPIO-1.25micL 0 500 1000 1500 2000 2500 3000 3500 15min 30min 45min NOSPIO Sample1 Sample2 Sample3
  7. 7. Dosage at 2.5micL 0 500 1000 1500 2000 2500 3000 3500 15min 30min 45min Sample1 Sample2 Sample3
  8. 8. Dosage at 5micL 0 500 1000 1500 2000 2500 3000 15min 30min 45min Sample1 Sample2 Sample3
  9. 9. Dosage at 10micL 0 200 400 600 800 1000 1200 1400 1600 1800 15min 30min 45min Sample1 Sample2 Sample3
  10. 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. 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. 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. 13. Dosage at 1.25micL 0 5 10 15 20 25 30 30min 180min Sample1 Sample2 Sample3
  14. 14. Dosage of 2.5micL 0 5 10 15 20 25 30 30min 180min Sample1 Sample2 Sample3
  15. 15. At 5micL 0 5 10 15 20 25 30 30min 180min Sample1 Sample2 Sample3
  16. 16. At 10micL 0 5 10 15 20 25 30 30min 180min Sample1 Sample2 Sample3
  17. 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. 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. 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. 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. 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. 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.

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