Peroxidetect kit

MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Phenol red-free DMEM/F-12 media, horse serum, penicillin/streptomycin, antibiotic/antimycotic, human insulin (Novolin R), epidermal growth factor, trypsin-EDTA (10X), Hanks Balanced Salt Solution (HBSS), and Phosphate Buffered Saline (PBS) were purchased from Invitrogen (Carlsbad, CA, USA). Cholera toxin was purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA). The CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay was purchased from Promega (Madison, WI, USA). The Bromodeoxyuridine Cell Proliferation (Chemiluminescent) Assay kit was purchased from Cell Signaling Technology (Danvers, MA, USA). The EpiQuik 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) was purchased from EpiGentek (Farmingdale, NY, USA). The Qiagen Genomic-tip 20/G, Genomic DNA buffer set, and proteinase k were purchased from Qiagen (Germantown, MD, USA). benzo(a)pyrene (BaP), diallyl sulfide (DAS), PeroxiDetect^TM Kit, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from Cell Signaling (GAPDH (D16H11) Rabbit mAb; #5174S) and Abcam (DNA Polymerase β; ab26343).

Total lipids were extracted from trophozoites by 1:2 chloroform:methanol (v/v), as described [30 ]. Lipid peroxidation was determined in lipids using the Peroxidetect^TM kit (Sigma-Aldrich). Briefly, 100 μL of lipid extracts were incubated with 1 mL of Working Color Reagent at room temperature (RT) for 30 min and color intensity was measured by spectrophotometry at 560 nm. Standard curves were generated for each experiment using 0 to 16 nmol of ter-BuOOH (Sigma-Aldrich). Absolute methanol was used as a blank.

The H₂O₂ production was studied by culturing microorganisms in TMB-MRS agar plates. The plates were prepared with MRS agar supplemented with 3,3′,5,5′-Tetramethyl-Benzidine (TMB) (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 0.25 mg/mL and horseradish peroxidase to a final concentration of 0.01 mg/mL. After incubation at 37 °C in a 5% CO₂ atmosphere for 48 h, the plates were exposed to the air. Colonies able to produce H₂O₂ develop a color of blue or brown. According to the color intensity, the strains were classified as strong (blue), medium (brown), weak (light brown), or negative (white colonies) producers [38 (link)].
For quantitative H₂O₂ determination, overnight LAB cultures were harvested by centrifugation, cells were washed twice with PBS and resuspended in 500 µL Lysogeny broth (LB). After 4 h incubation with shaking at 200 rpm at 37 °C, the suspension was centrifuged 3 min at 7000 rpm and H₂O₂ concentration was measured in the resulting supernatant by the method based on oxidation of ferrous to ferric ion in the presence of xylenol orange (XO). PeroxiDetect^TM Kit (Sigma-Aldrich) was used following the manufacturer’s instructions.