Dimethylsulfoxyde dmso

PBMC from septic patients and healthy volunteers were isolated from 7 mL of blood freshly drawn on anticoagulated tubes with Lympholyte®-H Cell Separation Media (Cedarlane, Burlington, Canada) centrifugation. PBMC were immediately cryoconserved after isolation in Roswell Park Memorial Institute medium 1640 (RPMI) complemented with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and glutamine (Eurobio Abcys, Courtaboeuf, France) containing 40% AB human serum (SAB) and 10% dimethylsulfoxyde (DMSO) (Sigma-Aldrich, Saint-Louis, MO).

UPLC-MS grade acetonitrile (ACN) and methanol (MeOH) were ordered from Biosolve (Dieuze, France). Ethyl 3-aminobenzoate methanosulfonate, dimethylsulfoxyde (DMSO) (purity ≥99.7%), ammonium hydroxide (NH₄OH) (purity ≥99%), and acetic acid (AA) (purity ≥99%) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ammonium acetate (NH₄Ac) (purity ≥99%) and heptane (purity ≥90%) were supplied by Merck-Millipore (Saint-Quentin en Yvelines, France). Ammonium formate (NH₄FA) (purity ≥99%) was acquired by Biosolve Chimie (Dieuze, France). Formic acid (FA) (purity 98%), isopropanol (IPA) and HPLC water were ordered by Fisher (Illkirch, France). Ammonium bicarbonate (NH₄HCO₃) (purity 99%) and sodium hydroxide were obtained by Fluka (Steinheim, Germany).
Labelled (¹⁵N, ¹³C, D) or unlabelled analytical standards (IS–S1 Table) (purity ≥97%) were purchased from Sigma-Aldrich, Fluka, CliniSciences (Nanterre, France), Chemservice (Dallas, United States), CDN isotopes (Quebec, Canada), or Merck-Millipore.
Stock solutions of individual standards at concentrations of 0.1, 0.2, or 1.0 mg/mL were prepared in ACN, H₂O, ACN/H₂O, DMSO, or MeOH according to their solubilities, and were stored at -18°C until use. Working standard mixtures were prepared by diluting the stock solutions with ACN.

Female BALB/c mice (n = 6–8 mice per group), purchased from Charles River Breeding Laboratories (L’Arbresle, France), were used for all experiments. Mice were housed in a ventilated cage system. The protocol was approved by the Ethics Committee on Animal Experimentation of the Pays de la Loire (accreditation number: 9456). Mice were sensitized on days 0, 7, 14, and 21 by percutaneous application of 500 μg of crude extract of Dermatophagoides farinae (Der f, Stallergenes Greer, Antony, France) diluted in 20 μl of dimethyl sulfoxyde (DMSO) (Sigma-Aldrich, Saint Louis, Missouri, USA) on the ears, without any synthetic adjuvant. They were challenged intranasally with 250 μg of Dermatophagoides farinae (Der f, Stallergenes Greer, Antony, France) in 40 μl of sterile phosphate buffered saline (PBS) on day 28 to induce AHR and again on days 29, 30, 35, 36, and 37 to enhance AHR. Mice were sacrificed on day 38 (Figure 1A).

Lyophilized synthetic PhlTx1 wild-type and variant forms (purity rate > 97%) were dissolved in appropriate solutions to give adequate stock solutions. All compounds used as reference molecules to pharmacologically validate the current flowing through each channel subtype, as indicated in Table 3, were purchased from Sigma-Aldrich and Smartox Biotechnology (Saint-Egrève, France).
The toxins were dissolved in distilled water to give a 100 µM stock solution, and the chemicals were dissolved in 100% dimethylsulfoxyde (DMSO, Sigma-Aldrich) to give a 10 mM stock solution. Just prior to experiments, successive dilutions were performed in the appropriate standard physiological medium (supplemented with 0.1% bovine serum albumin (BSA, Sigma-Aldrich) for toxins to avoid plastic adherence, and with 0.3% DMSO for chemicals to maintain solubility), to give the compound final concentrations indicated in the text.