A6283

The analysis of VFA (acetic, propionic, butyric, isobutyric, valeric, and isovaleric) of the samples of ruminal fluid was performed by gas chromatography using a flame ionization detector. A volume of 4 mL of diluted ruminal fluid mixed with 1 mL of a solution of 20 g/L of 4-methylvaleric acid as an internal standard, in 0.5 N HCl, was centrifuged (15,000× g for 15 min at 4 °C) to separate the liquid phase from the feed residuals. After, the liquid phase was microfiltered (premium syringe filter regenerated cellulose, 0.45 µm, 4 mm, Agilent Technologies, Madrid, Spain), and 0.5 µL of liquid phase was directly injected in the chromatograph (Agilent 6890 N) using a capillary column (30 m × 530 um; 1-µm particle size; HP- FFAP, Agilent, Spain) and kept at 300 °C in the injector with a hydrogen flow 40 mL/min, air flow 400 mL/min, and make up (nitrogen) 25 mL/min flow. The injection loop was 20 µL. The individual VFA were identified using a standard solution of 4.50 mg/mL of acetic acid, 5.76 mg/mL of propionic acid, 7.02 mg/mL of butyric acid and isobutyric acid, 8.28 mg/mL of valeric acid, and isovaleric acid in 0.1N H₂SO₄ (A6283, P1386, B103500, I1754, 240370, 129542, respectively; Sigma-Aldrich, Madrid, Spain). The quantification was performed using an external calibration curve based on the standards described above. Data were expressed in mmol/100 mmol.

A total of 12,000 cells per well were seeded in a 96-wells plate. Two days after seeding, media was replaced and cells were treated with increasing concentrations of a specific LPP for 72h. Then, cells were fixed with 1% trichloroacetic acid (#T9159-100G; Sigma-Aldrich) at 4⍰°C for 30 min, washed 3 times with distilled water and stained with 0.057% SRB (#S1402-5G; Sigma-Aldrich) in 1% acetic acid (#A6283, Sigma-Aldrich) solution at RT for 30 min. Following staining, cells were washed 3 times with 1% acetic acid and air-dried overnight. The protein bound dye was dissolved in 10⍰mM Tris base solution and the absorbance was measured at 565⍰nm using a microplate reader (SpectraMax i3x).

For observing the histopathological changes in nasal mucosa after CGA or Dex intervention, a 4-cm-thick mouse nasal mucosa was fixed in 4% paraformaldehyde (P6148, Sigma–Aldrich, U.S.A.), dehydrated by ethanol (E7023, Sigma–Aldrich, U.S.A.), blocked in paraffin (327204, Sigma–Aldrich, U.S.A.), and sliced. Then, the sliced mouse nasal mucosa was soaked in xylene (95670, Sigma–Aldrich, U.S.A.) for dewaxation. Gradient ethanol 100, 90, 80, and 70% was used to rehydrate mouse nasal mucosa slices. Mouse nasal mucosa slices were stained by Hematoxylin (H3136, Sigma–Aldrich, U.S.A.) for 15 min, differentiated by being soaked in 5% acetic acid (A6283, Sigma–Aldrich, U.S.A.), and immersed in distilled water for developing blue. Next, mouse nasal mucosa slices were stained by Eosin (E4009, Sigma–Aldrich, U.S.A.) for 10 min. Gradient ethanol 70, 80, 90, and 100% was used to dehydrate mouse nasal mucosa slices. After soaking in xylene twice, mouse nasal mucosa slices were sealed by Canada balsam (60610, Sigma–Aldrich, U.S.A.) and observed using an optical microscope (M3800, Fisher Scientific, Waltham, MA, U.S.A.).

Sirius red is a polyazo dye which is specifically used for staining collagen. The stain dyes collagen bright red, leaving muscle fibers, cytoplasm, and red blood cells a lighter yellow/red color. Picrosirius red differs from sirius red staining with the addition of picric acid which prevents the indiscriminate staining of noncollagenous structures by sirius red.
Sections were fixed in neutral buffered formalin for 30 minutes. Slides were rinsed in dH₂O and stained with Weigert's hematoxylin for 8 minutes. Sections were washed in 3 changes of water followed by staining with picrosirius solution for one hour. Picrosirius solution was made up of 0.5 g sirius red F3B (CI35780, Sigma-Aldrich, USA) in 500 ml saturated aqueous picric acid (197378, Sigma-Aldrich, USA). Sections were washed in 0.5% acetic acid (A6283, Sigma-Aldrich, USA) in dH₂O twice for 5 minutes each. Sections were then dehydrated in changes of ethanol (70%, 95%, and 100%) and then cleared in xylene (296325, Sigma-Aldrich, USA), and cover slips were mounted with mounting media (DPX, 06522, Sigma-Aldrich, USA).

Cells were seeded at a density of 10⁴ per well in 96 well plates overnight and incubated with cell‐permeant 1 μM 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA; D399, Thermo Fisher Scientific). Microplates were analyzed with a BMG Polarstar fluorescence spectrometer (ex 485‐12/em 520). Cell cultures were normalized to cellular protein as previously described.²⁴ Cell cultures were fixed with cold 10% (w/v) trichloroacetic acid (T4885‐1KG, Sigma‐Aldrich) and 0.004% (w/v) Sulforhodamine B (230162‐5G, Sigma‐Aldrich) solution, washed twice with 1% (w/v) acetic acid (A6283, Sigma‐Aldrich) and analyzed by fluorescence spectrometry (ex 544/em 590).

The AD-MSC cultures at the late passages were treated with 5.5 μg/mL Colcemid for 20 minutes in 37°C to stop cell divisions. The next step was hypotonization in 0.075 M solution of KCl (MERCK, 1.04936.1000) for 20–40 minutes, in 37°C. Then cells were fixated with Carnoy's mixture precooled to −20°C (methanol (MERCK, 106009.2511) with acetic acid (SIGMA, A6283) in proportions 3 : 1). Fixated cells were scraped and in small volume of fixation buffer (20 μL) delocalized on slides. The slides were stained according to modified Wang and Fedoroff's method (GTG: G-bands by Trypsin using Giemsa-stain) for bands visualization. This method embraced digestion with 0.1 M HCl (SIGMA, 115752837) for 2-3 seconds, incubation in 2x SSC solution (Standard Sodium Citrate, SIGMA, S4641) at pH 7, for 15 minutes, in 60°C, and staining with 0.25% Wrights solution (SIGMA, 861375) dissolved in phosphate buffer (SIGMA) at pH 6.8, in proportions 1 : 3 for 2-3 minutes. After every group of steps of staining, slides were washed under tap water and dried by warm airflow.
The stained slides were digitalized then observed metaphases were photographed and analyzed in 250x enlargement by IKAROS software of Meta Systems, system for picture analysis.
Karyotyping was done according to International System for Human Cytogenetic Nomenclature's guidelines (ISCN, 2013).