Positive Control. • 1.5 mL digitonin horseradish peroxidase conjugate in StabilZyme SELECT Stabilizer as a buffer solution. The positive control is 10x concentration of Detector Reagent
Positive Control. A positive control was used for all PM10 exposures in order to verify that the experimental set up was functioning and in order to establish consistency between different exposures. Standard Reference Material 1648a of the National Institute of Standards and Technology (NIST) was used as a positive control. NIST SRM1648a is characterized as atmospheric particulate matter collected in an urban area and represents in the current experimental series ambient particulate matter with an established oxidative potential for our model. It contains several metals, predominantly; iron, aluminium, and potassium, as well as high concentrations of silicon dioxide and sulphate [301]. NIST SRM1648a was weighed and suspended in chelexed water containing 5% to a final concentration of 500ug/ml, the addition of methanol aimed to facilitate suspension. In order to verify that this does not affect the redox potential of the controls, ‘blank NIST’ controls were also run, these contained the 5% chelex water used for the NIST dilutions and consistently it was shown that this very small methanol concentration had no effect. The resulting NIST suspension was vortexed vigorously for 5 minutes and then sonicated for 30 seconds at an amplitude of 15 microns. Aliquots of 1ml were stored in Simport ´O´ ring microtubes at -80 °C. Before exposure these stocks were diluted to 55.56 μg/ml through a two step dilution. This concentration was chosen because on the day of the exposure 50μl of the 2mM RTLF stock is added to 450μl of each 55.56μg/mL PM suspension, resulting in concentrations of 200μM and 50μg/mL respectively. For the dilution, firstly 1ml of the 500ug/ml aliquots were transferred in a falcon tube containing 2,333mls chelexed water (5% methanol) and were sonicated for 30 seconds at 15microns. The resulting 150μg/ml concentration was diluted to 55.56µg/ml by the addition of 555μl of the aforementioned solution in eppendorfs containing 944μl chelex treated water. NIST controls were run concurrently to the PM exposures and the antioxidant quantification methods. They resulted in consistent antioxidant depletion that was statistically significant when compared to the background oxidation controls (Figure 18).
