Coomassie plus assay reagent

Two and a half million cells were centrifuged at 2000 rpm (450 xg) for 5 minutes and washed with 1 mL PBS. The pellet was resuspended in 500 µl PBS and homogenized for 2 minutes. The sample was then centrifuged at 10,000 rpm at 4°C for 5 minutes, and the supernatant was aliquoted in an eppendorf tube and stored at -80°C until use. The levels of reactive oxygen and nitrogen species (i.e. hydrogen peroxide, peroxyl radical, nitric oxide and peroxynitrite anion), generally referred to as “ROS”, were then measured in these cell homogenates using the OxiSelect In Vitro ROS/RNS assay kit (Cell BioLabs, San Diego, CA). The fluorescence intensity was detected at 480 nm excitation/530 nm emission using a SpectraMax i3X fluorometric plate reader (Molecular Devices). Protein concentrations were also measured with the Coomassie Plus Assay Reagent (Thermo Fisher Scientific, Rockford, IL) and were used for normalization. Therefore, ROS levels are expressed as Relative Fluorescence Unit (RFU) per μg of protein.

Specimens isolated from the TZ and EC were weighed and transferred to glass vials containing PBS for homogenization. The volume of PBS used for each sample was calculated to yield 200 mg of tissue in 1 ml of PBS. Homogenates were centrifuged at 10,000 rpm for 5 min at 4 °C, after which the supernatant was transferred to a new eppendorf tube, then snap-frozen in liquid nitrogen and stored at − 80 °C until use. The levels of reactive oxygen and nitrogen species (generally referred to as “ROS”) in these tissue homogenates were measured using the OxiSelect In Vitro ROS/RNS assay kit (Cell BioLabs, San Diego, CA) according to the manufacturer’s protocol. The assay employs a proprietary quenched fluorogenic probe, dichlorodihydrofluorescin DiOxyQ which becomes rapidly oxidized to the highly fluorescent 2′, 7′-dichlorodihydrofluorescein by hydrogen peroxide, peroxyl radical, nitric oxide and peroxynitrite anion. Therefore, the fluorescence intensity measured at 480 nm excitation/530 nm emission using a fluorometric plate reader is proportional to the total levels of ROS/RNS within the samples. Protein concentrations were measured with the Coomassie Plus Assay Reagent (Thermo Fisher Scientific, Rockford, IL) and were used for normalization. ROS/RNS levels are expressed as Relative Fluorescence Unit (RFU) per μg of protein.

Whole protein was isolated from frozen cell pellets using RIPA buffer and protein concentration of lysates was determined by Bradford assays (Pierce™ Coomassie Plus Assay Reagent, Thermo). 25–40 µg of total protein lysates were separated on 12% SDS-PAGE gels and transferred to PVDF membranes (Merck) by using the turbo semi-dry blotting system (BioRad). Membranes were blocked with 5% BSA solution and incubated with primary antibodies (NGFR, clone D4B3, rabbit; GAPDH, clone D16H11, rabbit (#5174) and β-Tubulin, clone 9F3 all from Cell Signaling Technology, Germany; all diluted 1:1000) overnight at 4 °C. For signal detection membranes were washed twice with PBS-Tween20 (0.1%) and incubated with a horse radish peroxidase (HRP)-coupled secondary antibody (goat anti-rabbit IgG, Cell signaling) for 1 h at RT and analyzed with an automated imaging system (Vilber).