ROS levels were estimated using DCFH-DA (Sigma), which measures
intracellular generation of hydrogen peroxide, a procedure for estimating
ROS. DCFH-DA passively enters the cell, where it reacts with ROS to
form the highly fluorescent compound, dichlorofluorescein (DCF). Approximately,
0.3 × 104 cells were seeded and treated with AgNPs
at different concentrations for 24 h and 5 mM NAC was added 2 h prior
AgNP treatment wherever mentioned to inhibit ROS. Following exposure
to AgNPs, the cells were washed with PBS and then incubated in 100
μL of working solution of DCFH-DA (2 mM DCFH-DA stock solution
was diluted to yield a 20 μM working solution) at 37 °C
for 30 min. The fluorescence was measured at 485 nm excitation and
530 nm emission using a microplate reader (Fluoroskan Ascent).37 (link)To measure peroxidase enzyme activity,18 (link) 0.05 M pyrogallol was added to 100 μL
of the protein lysate. The reaction was started by adding 1% H2O2. Change in absorbance after every 30 s interval
for 3 min was observed in Multiskan Microplate Spectrophotometer at
420 nm. The enzyme activity was measured as a change in absorbance/min/mg
of protein. Pyragallol in the presence of H2O2 is oxidized to purpurogallin, a colored derivative, by the peroxidase
enzyme. The values were expressed as a fold change with respect to
control.
intracellular generation of hydrogen peroxide, a procedure for estimating
ROS. DCFH-DA passively enters the cell, where it reacts with ROS to
form the highly fluorescent compound, dichlorofluorescein (DCF). Approximately,
0.3 × 104 cells were seeded and treated with AgNPs
at different concentrations for 24 h and 5 mM NAC was added 2 h prior
AgNP treatment wherever mentioned to inhibit ROS. Following exposure
to AgNPs, the cells were washed with PBS and then incubated in 100
μL of working solution of DCFH-DA (2 mM DCFH-DA stock solution
was diluted to yield a 20 μM working solution) at 37 °C
for 30 min. The fluorescence was measured at 485 nm excitation and
530 nm emission using a microplate reader (Fluoroskan Ascent).37 (link)To measure peroxidase enzyme activity,18 (link) 0.05 M pyrogallol was added to 100 μL
of the protein lysate. The reaction was started by adding 1% H2O2. Change in absorbance after every 30 s interval
for 3 min was observed in Multiskan Microplate Spectrophotometer at
420 nm. The enzyme activity was measured as a change in absorbance/min/mg
of protein. Pyragallol in the presence of H2O2 is oxidized to purpurogallin, a colored derivative, by the peroxidase
enzyme. The values were expressed as a fold change with respect to
control.