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2 protocols using salivette roll shaped polyester swabs

1

Saliva Collection and Analysis Protocol

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Lactate (TraceCERT, 11.10 ± 0.02 mM), 9-chloromethyl-anthracene (purity ≥98%), tetra-n-butylammonium bromide (purity ≥98%), triethanolamine (purity ≥99%), uric acid (purity ≥99%), sodium hydroxide (purity ≥98%, pellets anhydrous), and hydrocortisone (purity ≥98%) were purchased from Sigma Aldrich (Milan, Italy).
8-isoPGF and 8-isoPGF-d4 were purchased at purity ≥99% from Cayman Chemical (Ann Arbor, Michigan, USA).
Phadebas® Alpha-Amylase Test was provided by Magle AB Life Sciences (Sweden).
TNF-α was quantified using the Quantikine® ELISA human TNF-α immunoassay (Cat. No. DTA00D) from R&D Systems (United States). Acetonitrile, methanol, and water at LC-MS grade (purity ≥99.9%) were from Fluka (Milan, Italy). Type I ultrapure water (18.2 MΩ-cm) for ELISAs was obtained using the Elga PURELAB Classic system (France).
All the liquid solutions and saliva samples were stored in sterile polypropylene containers from Eppendorf (Milan, Italy).
Phenex™-RC syringe filters (0.2 μm regenerate cellulose, 4 mm of diameter) were from Phenomenex (California, USA).
Salivette roll-shaped polyester swabs were purchased from Sarstedt (Nümbrecht, Germany).
A Macherey Nagel pehanon narrow range (6.0 < pH < 8.1) pH paper strips (Düren, Germany) with a resolution of 0.3 pH units was used to estimate the salivary pH.
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2

Salivary Biomarkers of Oxidative Stress

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Commercially available prostaglandin E 2 (PGE 2 ), F 2 -and E 2 -isoprostanes (IsoPs), 15-F 2t -IsoP and 15-E 2t -IsoP (purity ≥99%) were from Cayman Chemical (Michigan, USA).
Not commercially available F 4 -NeuroPs (20-F 4t -NeuroP, 20-epi-F 4t -NeuroP), F 2 -dihomo-IsoPs (ent-7(R,S)-7-F 2t -dihomo-IsoP, 17-F 2t -dihomo-IsoP) and F 2 -dihomo-IsoF (7(R,S)-ST-Δ 8 -11-dihomo-IsoF) were synthesized at the Institut des Biomolecules Max Mousseron (IBMM) (Montpellier, France) according to procedures reported elsewhere [41] (link)[42] (link)[43] (link)[44] (link).
All the solutions and saliva samples were stored in sterile polypropylene containers from Eppendorf (Milan, Italy).
Phenex™-RC syringe filters (0.2 μm regenerate cellulose, 4 and 15 mm of diameter) were from Phenomenex (California, USA).
The eVol® XCHANGE analytical syringe (20-500 μL) and microextraction by packed sorbent silica-C18 Barrel Insert and Needles (BINs) were purchased from SGE Analytical Science (Melbourne, Australia).
Salivette roll-shaped polyester swabs were purchased from Sarstedt (Nümbrecht, Germany).
A Macherey Nagel Pehanon narrow range (5.2 < pH < 8.1) pH paper strips (Düren, Germany) with a resolution of 0.3 pH units was used to estimate the salivary pH.
An Amicon Ultra-0.5 mL Centrifugal Filters (Darmstadt, Germany) with a cut-off of 3 kDa was used to purify saliva samples.
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