detection of production preliminary

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, we wanted to check if our SPIO would produce ROS.

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 .

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.

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)

Dosage of SPIO-1.25micL

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Dosage at 2.5micL

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Dosage at 5micL

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Dosage at 10micL

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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.

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.

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

Dosage at 1.25micL

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30min 180min

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Dosage of 2.5micL

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At 5micL

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At 10micL

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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 ??

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 37oC and 5% Co2. They were read for viability using an Elisa at 570 nm

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.

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.

Viability values with alamar blue

0.5850.59

0.5950.6

0.6050.61

0.6150.62

0.6250.63

1.25mL 2.5mL 5mL 10mL Untreated

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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.