Detection of production of reactive oxygen
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- The document summarizes experiments showing that superparamagnetic iron oxide particles (SPIO) taken up by macrophages produce reactive oxygen species (ROS). - When SPIO were added to macrophages in culture, the macrophages phagocytosed the particles. This was followed by the release of extracellular superoxide and later intracellular hydrogen peroxide, as measured using luminescence and fluorescence assays. - ROS production occurred for all SPIO doses tested. However, macrophage viability was not affected, as assessed using an Alamar Blue assay, indicating ROS production did not lead to cell death over 24 hours.
Detection of production of reactive oxygen
- 1. Detection of production of Reactive Oxygen Species by 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 the 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. • 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 FDIO. 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 internalized 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-1.25mL 0 500 1000 1500 2000 2500 3000 3500 15min 30min 45min NOSPIO Sample1 Sample2 Sample3
- 7. Dosage at 2.5mL 0 500 1000 1500 2000 2500 3000 3500 15min 30min 45min Sample1 Sample2 Sample3
- 8. Dosage at 5mL 0 500 1000 1500 2000 2500 3000 15min 30min 45min Sample1 Sample2 Sample3
- 9. Dosage at 10mL 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 fluorometer.
- 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 extracellular 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. ∀•H2O2 was released by SPIO at all doses tested (1.25-10 ul/) ∀•Note untreated macrophages have a background fluorescence of < 2 AFU
- 13. Dosage at 1.25mL 0 5 10 15 20 25 30 30min 180min Sample1 Sample2 Sample3
- 14. Dosage of 2.5mL 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 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 extracellular 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. 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 treatment.
- 20. 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
- 21. Future plan • As ox-LDL is know to produce ROS, we would like to see the same effect in the presence of our SPIO.