Superoxide dismutase

A traditional approach based on cytochrome C reduction was employed to determine the rate of superoxide anion release in NHEK cells [60 (link)]. Both treated and untreated cells received 100 μL of cytochrome C and 100 μL of superoxide dismutase (all chemicals were given by Merck Life Science, Rome, Italy) for 30 min in an incubator. A spectrometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) was used to detect the absorbance in culture supernatants at 550 nm, and O₂ is expressed as the mean ± SD (%) of nanomoles of reduced cytochrome C per microgram of protein compared to the control (0 line) [45 (link)] of five independent experiments performed in triplicate.

Reagents FAD disodium
salt hydrate (FAD-Na₂), β-nicotinamide adenine dinucleotide
2′-phosphate reduced tetrasodium salt (NADPH), Roswell Park
Memorial Institute (RPMI) 1640 medium with l-glutamine and
sodium bicarbonate, phosphate-buffered saline (PBS), lithium dodecyl
sulfate (LiDS), CaCl₂, sodium chloride, HEPES buffer, sodium
dihydrogen phosphate (NaH₂PO₄·H₂O), phenylmethylsulfonyl fluoride, glycerol, pepstatin, leupeptin,
SigmaFast protease inhibitor cocktail tablets EDTA-free, cytochrome
c from equine heart, hemin, superoxide dismutase, Ampliflu Red (Amplex
Red) and horseradish peroxidase, Triton X-100 reduced, phorbol myristate
acetate (PMA), VAS2870 and VAS3947, kynuramine, and benzylamine were
purchase from Sigma-Aldrich. MCLA was purchased from MedChemExpress.
Cholesteryl hemisuccinate (CHS), dodecyl-β-d-maltopyranoside
(DDM), β-octylglucoside (OG), and n-dodecyl-phosphocholine
(Fos-Choline-12) were purchased from Anatrace. Fetal bovine serum
(FBS), FreeStyle and DMEM (cat. no. 31966047) media, penicillin G,
and streptomycin were purchased from Invitrogen (Carlsbad, CA).

Lettuce (Lactuca sativa L.) types romaine, iceberg, butterhead, and green and red leaf were obtained as mature heads from distributors. Bundled spinach (Spinacia oleracea L.) and bagged baby spinach were obtained from retail stores. Lettuce heads and bundled spinach were separated into young and middle leaves and the very outer leaves were discarded. Young and middle leaves corresponded approximately to the 6th (partially expanded) and 12th (fully expanded) positions of the leaves starting from the meristem rosette toward the outer part of the lettuce head, respectively.
Two middle leaves and three young leaves from the same mature lettuce heads or spinach bundles were crushed to a fine paste with a mortar and pestle and as separate composite samples based on the age of the leaves. The resulting lysates were centrifuged immediately at 12,000× g at 4 °C for 5 min to pellet cellular debris. The supernatant was filter-sterilized by passing through a 0.45-μm pore-size PVDF (polyvinylidene difluoride) filter (Fisher Scientific, Waltham, MA, USA) and immediately used for inoculation with EcO157 and measurement of H₂O₂ and carbohydrate concentrations. This method for lysate production was used successfully in our previously published transcriptome study in which EcO157 was exposed to romaine lettuce cellular contents [5 (link)]. For experiments on the remediation of oxidative stress, catalase and superoxide dismutase (Sigma Aldrich, St-Louis, MO, USA) were added to lysates at 150 units/mL and 300 or 3000 units/mL, respectively. All lysates were used immediately for bacterial density measurements and chemical analyses.
The lysates were sampled for EcO157 concentration at 0, 1, 2, 4, and 5 h after inoculation and incubation at 100 rpm at 28 °C to mimic temperature abuse. Plate counts were performed after dilution plating of lysate aliquots onto Luria–Bertani agar containing 100 μg/mL rifampin and plate incubation overnight at 37 °C. Two replicates of composite lysate samples were tested using two different lettuce heads, spinach bundles, or bags of baby spinach. Each lettuce type and spinach were tested in two replicate experiments with plant material obtained from suppliers on separate days. Data from plate counts were used to calculate bacterial density change, here defined as the increase in cell concentration from 0 h at the time of inoculation to 5 h post-incubation.