Amplex Red

We measured the total cellular cholesterol using the Amplex Red cholesterol assay kit (Invitrogen, Waltham, MA, USA) in accordance with the manufacturer’s protocols. Briefly, prepared cultured cells were harvested and kept at −70 °C. The frozen cells were incubated with reaction buffer for 30 min on ice, and then sonicated to disrupt the cellular membrane. Samples (50 µL) were mixed with equal volume of Amplex Red reagent containing HRP, cholesterol oxidase, and cholesterol esterase, and then the mixture was reacted for 30 min at 37 °C. The fluorescence intensity was measured using a fluorescence microplate reader (BioTek, Winooski, VT, USA) at ex/em = 530/590. The cholesterol levels were normalized using protein concentration determined by Bradford assay.

Myocardial superoxide generation was measured via in situ dihydroethidium (DHE, Thermo Fisher Scientific, D11347) staining. Briefly, NRCMs were incubated with DHE (5 μM) in a humidified chamber at 37 °C, followed by PBS washing. The staining (red) images were captured with the microscope (EVOS, Thermo Fisher Scientific, MA, USA). The fluorescence intensity of DHE staining was measured using the ImageJ software (version 1.8.0), and the results were expressed as fold change against the corresponding controls.
Amplex Red assay (Invitrogen) was used to detect hydrogen peroxide levels in mouse hearts and NRCMs according to the manufacturer’s protocol. For mouse hearts, fresh frozen tissue (30–60 μg) was incubated in Amplex Red working solution (containing 100 μM Amplex Red reagent and 1 U/mL horseradish peroxidase) in HEPES buffer for 30 min at 37 °C. The supernatant was then collected, and the fluorescence was measured at 530 nm excitation wavelength and 590 emission wavelength using a Mithras LB 940 reader (Berthold Technologies). All procedures were protected from light, and a standard curve using fresh H₂O₂ was generated for comparison.
For the detection of hydrogen peroxide levels in NRCMs cells. Equal amounts of cells seeded in 96-well plates were incubated with Amplex Red working solution (containing 50 μM Amplex Red reagent and 0.1 U/mL horseradish peroxidase) for 30 min at 37 °C. Fluorescence was measured at 530 nm excitation wavelength and 590 nm emission wavelength using a Mithras LB 940 reader (Berthold Technologies). All procedures were protected from light, and a standard curve using fresh H₂O₂ was generated for comparison.
The production of ROS was measured using the 2’,7’-dichlorodihydrofluorescein-diacetate (DCFH-DA, Sigma-Aldrich, D6883). For the in vivo study, 5 μg tissue homogenate was incubated with 4 μM DCFH-DA for 10 min at 37 °C, and for the in vitro study, equal amounts of cells cultured in 96-well plates were incubated with 10 μM DCFH-DA for 20 min at 37 °C, protected from light. The fluorescence was then measured at 485 nm excitation wavelength and 535 nm emission wavelength using a Mithras LB 940 reader, and the data were analyzed with MikroWin 2010 software (Berthold Technologies).

Nasal secretions were collected by nasal lavage from control subjects with blowout fractures and patients with CRS. Briefly, 5 mL of phosphate-buffered saline (PBS) was flushed through a catheter into each nasal cavity. Thereafter, lavage fluid was collected in a sterile tube. The supernatant obtained after centrifuging the pooled lavage fluid was stored in a deep freezer until H₂O₂ analysis.
The concentration of H₂O₂ in nasal secretions was evaluated using the Amplex Red hydrogen peroxide assay kit (Invitrogen, CA, USA). In brief, a buffer solution in 100 μM Amplex Red reagent was added to nasal secretions collected by nasal lavage and then mixed with 0.2 U/mL horseradish peroxidase (HRP), adjusted to a final volume of 100 μL. The reaction of Amplex Red reagent with H₂O₂ resulted in the production of red fluorescent resorufin, which was evaluated using a fluorescence plate reader. BCA protein assay kit (Thermo Fisher Scientific, Mass, USA) was employed to measure the concentration of total protein contained in nasal lavage fluid.

Hemocytes were plated in 96-well plates containing treatments in their respective culture mediums in a total volume of 70–100 µL with 5 replicates of each treatment. The plates were sealed with sealing tape and incubated for 3 h at 20 °C. Extracellular hydrogen peroxide was quantified using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen™, Waltham, MA, USA, catalog no. A22188). A 100 μM Amplex^® Red reagent and 0.2 U/mL HRP working solution in 1× reaction buffer were prepared according to the manufacturer’s protocol. Hydrogen peroxide standards were prepared in 1× reaction buffer with concentrations between 0 and 10 µM. Plates were centrifuged using ThermoScienfic™ Megafuge 16R centrifuge at 1000× g rcf for 3 min, and 50 µL of the supernatant was transferred to a new 96-well plate. The working solution was transferred in 50 µL volume to each well containing the supernatant and the hydrogen peroxide standards, then incubated for 30 min in the dark. Absorbance was measured using a spectrophotometer (BioTek™, Winooski, VT, USA, PowerWave XS2) at λ = 560 nm and the results were normalized to the absorbance of the culture medium, expressed as relative to the control group with no pesticide exposure.