Tbars tca method assay kit

C. albicans cells from overnight culture were grown in YPD medium to exponential phase. Cells were harvested, washed twice with 12.5 mM sodium acetate and resuspended in 12.5 mM sodium acetate to 4 × 10⁶ cells/ml. A total of 100 ml of cells were treated with AMPs (P-113, 1 μM; P-113Du, 0.1 μM; P-113Tri, 0.05 μM) in 12.5 mM sodium acetate and incubated at 37 °C for 2 h. Subsequently, cells were harvested, washed twice with PBS and mixed with ice-cold protein extraction buffer (5 mM potassium phosphate, 0.9% (w/v) sodium chloride, and 0.1% (w/v) dextrose, pH 7.4) and 0.3 g acid-washed glass beads (Sigma-Aldrich, 425~600 μm). Cells were disrupted by vortexing for 20 s and immediately cooled on ice for 20 s. The process was repeated eight times. Cell lysates were centrifuged (13000 × g, 4 °C, 15 min) and the supernatants were collected. Total protein was quantified by Bradford protein assay at 600 nm using an iMark microplate absorbance reader (Bio-Rad). The MDA assay was performed using the TBARS (TCA method) Assay Kit (Cayman Chemical), following the manufacturer’s instruction. All experiments were repeated at least three times.

Plasma malondialdehyde concentrations and total antioxidant capacity were used for the evaluation of the feto–placental redox status. The quantification of malondialdehyde was performed using a colorimetric kit (TBARS, TCA method, Assay Kit, Cayman Chemical Company, Ann Arbor, MI, USA), according to the manufacturer’s instructions. The absorbance was read at 540 nm with a microplate reader (Perlong DNM-9602, Nanjing Perlove Medical Equipment Co. Ltd., Nanjing, China). The assessment of total antioxidant capacity in plasma was performed using a colorimetric Antioxidant Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA), according to the instructions of the manufacturer. The absorbance was read at 405 nm with the microplate reader. Both assay kits have been previously used with ovine plasma [28 (link)].

To determine the extent of lipid peroxidation, the levels of MDA were measured. C. albicans cells were treated with or without LL-37 (8 μg/mL) for 30 min. Subsequently, cells were harvested, washed twice with PBS and resuspended in PBS containing acid-washed glass beads. The cells were vortexed for 30 s and cooled on ice for 30 s, and this process was repeated 10 times. Cell debris was removed by centrifugation at 4 °C for 10 min, and the supernatant was collected. Next, lipid peroxidation was measured using the TBARS (TCA method) Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. In this assay, TBARS formed by the reaction of MDA and thiobarbituric acid (TBA) under high temperature (90–100°C) and acidic conditions was measured colorimetrically at 530 nm.

HT-1080 cells were seeded into 150 mm dishes with a density of 2 million cells per dish. After 48 h of growth, cells were treated with RSL3 and Ferrostatin-1 or Turofexorate/Fexaramine for 2.5 h before they were harvested using 0.05% Trypsin-EDTA (Thermo Fisher Scientific). Cells were counted and cell number was adjusted to the sample with the lowest cell count. TBARS Assay was performed according to the user manual of the TBARS (TCA Method) Assay Kit (Cayman Chemical, 700870). Malondialdehyde (MDA) levels were obtained by detecting fluorescence at 530 nm/550 nm in an EnVision 2104 Multilabel plate reader (PerkinElmer).

The TBAR level was evaluated using the TBARS (TCA method) Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA, No. 700870) with spectrophotometric analysis, according to the manufacturer’s instructions [30 (link)]. This kit measured lipid peroxidation by reacting MDA with TBA under high temperatures and acidic conditions to form an MDA–TBA adduct, after which absorbance was measured at 530–540 nm.