Total antioxidant capacity assay kit

RBC pellet was transferred to a clean tube with 1 * 10⁷ RBC/ml in 0.1 mol PBS (pH 7.4), snap frozen and stored at −80 °C until measurement. Samples were thawed on ice and the total antioxidant capacity assay kit (Abcam, UK) was applied to measure RBC total antioxidant capacity according to the manufacturers´ instructions.

All assays were analyzed on a Tecan Sunrise microplate reader. The markers of inflammation/oxidative stress that we measured were as follows: 8-hydroxy-2’-deoxyguanosine (8-OHdG) (Trevigen, Gaithersburg, MD), protein carbonylation concentration (OxiSelect Protein Carbonyl ELISA Kit, Cell BioLabs), small molecule antioxidant capacity (Total Antioxidant Capacity Assay Kit, Abcam), catalase activity (StressXpress Colorimetric Activity Kit, StressMarq Biosciences Inc), and ferritin (Ramco Laboratories, Stafford, TX) according to the manufacturers’ protocol.

The total anti-oxidant capacity was measured in HBMECs and OECs using a total antioxidant capacity assay kit (Abcam, Cambridge, UK) as per manufacturer’s instructions. In brief, cells grown under normal and experimental conditions were pelleted by trypsinisation which were then re-suspended with double distilled water (ddH₂O) before centrifugation for 4 min at 10,000 rpm to obtain the supernatant. 100 µL of working solution (the mixture of Cu²⁺ reagent and assay diluent) was then mixed with samples and standards and incubated at room temperature for 90 min before measuring total anti-oxidant capacity in a plate reader (FLUOstar Omega, BMG Labtech, Aylesbury, UK) at 570 nm.

To evaluate the antioxidant effect in both medium and neutrophils, we utilized the Total Antioxidant Capacity Assay Kit from Abcam (Cat. No. ab65329, Cambridge, UK) following the manufacturer’s protocol without the protein mask. The TAC was assessed in the culture media components: RPMI 1640 alone, AS alone, and RPMI 1640 + AS, following the manufacturer’s instructions for liquid samples. For the evaluation of TAC in neutrophils, we seeded 1 × 10⁶ neutrophils and followed the same protocol described in the section of NETosis assay for the controls and the combination of antioxidants GSH + NAC and ALL. After inducing NETosis, in all cases, the cell suspensions were washed twice with cold PBS 1× to remove the media. Subsequently, the samples were resuspended in 100 µL of ddH₂O containing 0.05% Triton X-100 (Cat. No. 85111, Thermo Fisher). The resulting homogenate was then incubated on ice for 10 min. After incubation, the samples were centrifuged at top speed for 5 min at 4 °C. The supernatant was collected, and the assessment was carried out according to the instructions provided in the kit. The output of the TAC assay was measured using a microplate reader (ALLSHENG, Hangzhou, China) at 570 nm.