Spectramax m5 plate reader

Luciferase assays were conducted using clear bottom white plates (Costar), with Promega's Dual-Glo(R) Luciferase Assay System according to manufacturer's recommended protocols following a 24-h incubation period at 37°C post-transfection. Briefly, Dual-Glo® Luciferase was added to each well (100 μL) and incubated at room temperature for 15 min. Firefly luminescence was measured using a M5 SpectraMax plate reader (Molecular Devices). Dual-Glo® Stop & Glo® was added (100 μL per well) to the 96-well plate and incubated for 15 min at room temperature before measuring Renilla luminescence.

Naphthotriazole, which was formed by the reaction of DAN and NO, was quantified using fluorescence spectroscopy [35 (link)]. Supernatants from platelets treated with Hb (3.0 μM) or thrombin (1 U/ml) were aliquoted into two equal parts; one part was treated with DAN (150 μM), and the other part was treated with DAN (150 μM) and copper acetate solution (150 μM). The reaction mixture was incubated for 30 min at night, and the reaction volume was adjusted to a final volume of 200 μl by adding 0.1 M NaOH. The OD was taken at an excitation wavelength of 375 nm and an emission wavelength of 450 nm using a Spectramax plate reader M5 (Molecular Devices, USA). NO release was quantified using a standard curve of NaNO₂ [36 (link)].

DAN reacts with NO to form napthotriazole (NAT) which can be quantified by fluorescence spectroscopy53 (link). In vitro nitrosylation of UCHL1 and TM was carried out by incubating equal volume of protein (20 μM) and GSNO solution (1 mM) in 1:50 molar ratio at 37 °C for 30 min. After nitrosylation, excess GSNO was removed by passing the sample through Bio-spin P6 column (Bio-Rad, USA). Nitrosylated protein sample was divided into two equal parts: one part was treated with DAN (150 μM) and other part was treated with DAN (150 μM) and copper acetate solution (150 μM). The reaction mixture was incubated for 30 min in dark and finally the volume was adjusted to 200 μl by adding 0.1 M NaOH. The fluorescence data was collected using Spectramax plate reader M5 (Molecular device, USA) with excitation wavelength at 375 nm and emission at 450 nm. GSNO was used to prepare standard curve for quantification of NO release.

An enzyme-linked immunosorbent assay (ELISA; Invitrogen, Carlsbad, CA, USA) was conducted to evaluate immunoglobulin (IgG) isotypes of anti-OVA antibodies in serum of immunized mice collected 6 and 10 days after tumor challenge. Briefly, 96-well ELISA plates were coated with 100 μg/mL OVA (Sigma-Aldrich) in PBS overnight at 4 °C. After washing with 0.05% Tween-20 in PBS (PBST), plates were blocked for 1 h in 10% FBS in PBS at 37 °C. After a thorough washing with PBST, diluted serum samples were added to each well and incubated with gentle shaking for 2 h. The plates were then washed again with PBST and incubated with horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG (1 μg/mL in 10% FBS) for 2 h with gentle shaking. Finally, the plates were washed and 3,3′,5,5′-tetramethylbenzidine substrate was added. After 10 min, the reaction was stopped with sulfuric acid (0.16 M) and absorbance was detected at 450 nm with a SpectraMax M5 plate reader (Molecular Devices).

The ROS level in C. elegans was measured as described [16 (link)]. Synchronized larvae were cultivated with PNS at 20°C for 10 days as an aged model, then the nematodes were collected and washed in M9 buffer for three times. Approximately 500 animals were homogenized in 500 μL PBS at 4°C. After centrifugation, the supernatant was collected and subjected to protein quantification. Then, 200 μg/mL lysate was transferred into a 96-well plate and incubated with 25 μM/well of DCFH-DA. The fluorescence was measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA), with an excitation 485 nm and an emission 535 nm. ROS level were measured every 10 min persistently for about 2.5 h.