Sodium dodecyl sulphate (sds)

Aliquots of CE were incubated for 10, 30, and 60 min at 60, 80, 90, and 100 °C as well as 15 min at 121 °C (autoclaving). In another batch, aliquots of CE were adjusted at pH 2.0, 4.0, 6.0, and 8.0, incubated for 3 h at room temperature. In addition, the effect of Triton X-100 (BDH Chemicals Ltd., Poole, UK), sodium dodecyl sulphate ((SDS) Sigma-Aldrich Corporation, St. Louis, MO, USA), and ethylenediaminetetraacetic acid ((EDTA) Sigma-Aldrich Corporation, St. Louis, MO, USA) at the final concentration of 1 mg/mL was evaluated. All experiments were run in triplicate using S. aureus ATCC1026, S.dysenteriae UTNFa37-1, K. cowanii B2Sh1, and E. coli ATCC25922 as indicator strains. The control for all experiments was the sterile MRS medium.

Phosphate-buffered saline (PBS) was from Avantor Performance Materials Poland S.A. (Gliwice, Poland). Arachidonate, collagen, and ADP were from Chrono-Log Corp. (Havertown, PA, USA). Dimethyl sulfoxide, cytochrome c, sodium dodecyl sulphate (SDS), Ellman's reagent (5,5′-dithiobis-2-nitrobenzoic acid, DTNB), glutathione (reduced), HCl, 2,4,6-trinitrobenzenesulphonic acid (TNBS), ethanol, ethyl acetate, guanidine hydrochloride, xylenol orange, Fe(NH₄)₂(SO₄)₂, and perchloric acid were from Sigma-Aldrich (St. Louis, MO, USA). Pierce™ BCA Protein Assay Kit was from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Fluorolabelled monoclonal antibodies (moAbs)—anti-CD61/PerCP, antiCD62/PE, PAC-1/FITC, isotype antibodies, and CellFix were from Becton Dickinson (San Diego, CA, USA).

All reactants were used without prior purification. 2,3:4,5-Di-O-isopropylidene-b-d-fructopyranose, also called diacetone-d-fructose (DAF, >98%), was obtained from Carbosynth (Compton, UK). Butyl acrylate (BA, 99.5%) was purchased from Quimidroga (Barcelona, Spain). Methacrylic anhydride (94%), triethylamine (NEt3, >99.5%), 4-dimethylaminopyridine (DMAP, >99%), potassium persulfate (KPS, >99%), sodium dodecyl sulphate (SDS, >98.5%), and NaHCO₃ (99.7%) were from Sigma-Aldrich (Steinheim, Germany). Solvents were purchased from Acros-Organics (Geel, Belgium) and Sigma-Aldrich.

All the reagents were of analytical grade and were used without further purification. AvanGRAPHENE, G powder with lamellar structural morphology comprising less than 6 layers with a thickness ≤2 nm, was supplied by Avanzare Innovación Tecnológica, SL (Logroño, Spain). Riboflavin (C₁₇H₂₀N₄O₆, Mw = 376.36 g mol⁻¹), sodium dodecylsulphate (SDS, NaC₁₂H₂₅SO₄, micellar critical concentration CMC = 8.2 mM, Mw = 288.38 g mol⁻¹), dodecyltrimethylammonium chloride (DTAB, CH₃(CH₂)₁₁N(CH₃)₃Br, CMC = 14.0 mM, Mw = 308.34 g mol⁻¹) and polyoxyethylene-23-lauryl ether (Brij L23, C₁₂H₂₅(OCH₂CH₂)₂₃OH, CMC = 91 μM, Mw = 1198.56 g mol⁻¹), were purchased from Sigma–Aldrich (Madrid, Spain). Hexadecyltrimethylammonium bromide (CTAB, C₁₉H₄₂BrN, Mw = 364.46 g mol⁻¹, CMC = 0.9 mM) was obtained from Merck (Barcelona, Spain). All the aqueous solutions were prepared using ultrapure water obtained from a Milli-Q system (Millipore, Milford, CT, USA).

McIntosh Red apples (Canada Fancy) were stored at 4°C in the dark for a maximum of two weeks. In order to prepare apple sections, the fruit was first chilled in a −20°C freezer for 5 minutes prior to being cut with a mandolin slicer to a uniform thickness of 1.20±0.14 mm, measured with a vernier caliper (Fig. 1 A-B). Only the outer (hypanthium) tissue of the apple was used. Slices containing visible ovary-core tissue were not used. The slices were then cut into 2.0×0.5 cm segments parallel to the direction of the apple pedicel (Fig. 1 C). Apple tissue was then decellularized by using a well-established protocol [41] (link) for removing cellular material and DNA from tissue samples while leaving behind an intact and three-dimensional scaffold. Individual apple tissue samples were placed in sterilized 2.5 mL microcentrifuge tubes and 2 mL of 0.5% sodium dodecyl sulphate (SDS) (Sigma-Aldrich) solution was added to each tube. Samples were shaken for 12 hours at 160 RPM at room temperature (Fig. 1 D). The resultant cellulose scaffolds were then transferred into new sterile microcentrifuge tubes, washed and incubated for 6 hours in PBS (Sigma-Aldrich) with 1% streptomycin/penicillin (HyClone) and 1% amphotericin B (Wisent). At this point, the samples were immediately used or stored in PBS at 4°C for no more than 2 week.

Next, 200 μg of proteins were diluted with the Laemmli buffer, boiled for 10 min, and loaded on SDS-8% polyacrylamide gel (Sodium Dodecyl Sulphate, Sigma-Aldrich, St. Louis, Missouri, USA). SDS-polyacrylamide gel electrophoresis was performed with a running buffer (Tris-HCl 25 mM, glycine 0.2 M, SDS 0.1%) at a 110 V constant voltage. Proteins were wet-transferred from the gel to a polyvinylidene fluoride membrane (PVDF Immobilon-P, Millipore) in a cold chamber (+4°C), for 1 h 30 min with a blotting buffer (glycine 0.15 M, Tris-HCl 25 mM, and methanol 10% in ultrapure water) and at a 250 mA constant current. The membrane was stained with Ponceau S solution (Sigma-Aldrich, St. Louis, Missouri, USA) to control the effective transfer of proteins, then washed five times with PBS-T (NaCl 130 mM, KCl 2.6 mM, Na₂HPO₄ 8.4 mM, KH₂PO₄ 1.4 mM, pH 7.4, and Tween 20 at 0.1%) and incubated with a blocking solution (5% milk in PBS-T) for 2 h, to mask aspecific sites of the membrane.

An aliquot of each sample was centrifuged (800× g, 10 min) and the sperm pellet was sonicated (28 kHz, 30 s) in the presence of the RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) and protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Following the second round of centrifugation (11,828× g, 4 °C, 10 min) and purification, the lysates were subjected to the quantification of malondialdehyde (MDA), considered to be the principal marker of lipid peroxidation (LPO).
The sperm lysates were pre-treated with 5% sodium dodecyl sulphate (Sigma-Aldrich, St. Louis, MO, USA) and subsequently exposed to 0.53% thiobarbituric acid (TBA; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 20% acetic acid (pH 3.5; Centralchem, Bratislava, Slovakia) under high-temperature conditions (90–100 °C) for 1 h. Afterwards, the samples were cooled down for 10 min and centrifuged (1750× g, 10 min). The supernatants (150 μL) were transferred into a transparent 96-well plate and the levels of MDA were assessed at 540 nm using the Glomax microplate spectrophotometer (Promega, Madison, WI, USA). MDA levels are expressed as μmol/L [71 (link)].