Lipid Peroxidation Assay

Lipid peroxidation was analyzed using a TBARS Assay Kit (Cayman Chemical, United States) by means of malondialdehyde (MDA) content (Poniedziałek et al., 2018). Following the incubation, cells were collected from 5 ml subsample of each culture by centrifugation, washed twice with BG-11 medium and incubated for 30 min at 21°C with gentle shaking on an orbital shaker with a cell-lysis buffer based on 1% Triton X-100 (Cayman Chemical, United States) supplemented with butylated hydroxytoluene (BHT) to prevent artificial lipid peroxidation. Following the incubation, samples were centrifuged (1,600 × g, 10 min, 4°C) to remove insoluble material. The protein content in supernatants (5 μl) was quantified with a Quick Start™ Bradford Protein Assay Kit (Bio-Rad) following the microassay procedure and using bovine serum albumin as a protein standard. The 100 μl of supernatants was transferred to a microcentrifuge tube and supplemented with 800 μl of thiobarbituric acid (TBA) to generate an MDA-TBA adduct. To accelerate this process, samples were incubated at 95°C for 60 min, placed on an ice bath for 10 min to inhibit the reaction and centrifuged (1,600 × g, 10 min, 4°C). The final product was measured colorimetrically at 532 nm using a Synergy HTX Multi-Mode Microplate Reader. The absorbance values were compared to a calibration curve prepared using the MDA standard (Cayman Chemical, USA) and calculated as nmol mL⁻¹.