Benzethonium chloride

Pro- and anti-inflammatory cytokines were quantified by ELISA assay. Briefly, the ileum section of were weighed and homogenized (100 mg tissue/ml buffer) in PBS containing 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO), phenylmethylsulfonyl fluoride 0.1 mM (Sigma- Aldrich, St. Louis, MO), benzethonium chloride 0.1 mM (Sigma-Aldrich, St. Louis, MO), EDTA 10 mM (Synth, São Paulo, São Paulo, Brazil), and aprotinin A 20 KIU (Sigma-Aldrich, St. Louis, MO). Suspensions were centrifuged at 3.000 g for 10 min 12 and the supernatants were collected for dosage of IL-1β, IL-6, IL- 10, IL-17, TNF-α, and INF-γ cytokine according to the R and D Systems, Inc. protocols. The absorbance was measured at 492 nm using a Microplate Reader Model 680 (BIO-RAD). Samples from six animals per group were collected for the ELISA assay, homogenized, and three technical replicates performed.

The colon was washed with PBS and placed in a buffer solution (1 ml/g) containing Tween-20 0.05% (Sigma-Aldrich, St. Louis, MO, USA), phenylmethylsulfonyl fluoride 0.1 mM (Sigma-Aldrich, St. Louis, MO, USA), benzethonium chloride 0.1 mM (Sigma-Aldrich, St. Louis, MO, USA), EDTA 10 mM (Synth, São Paulo, São Paulo, Brazil), and aprotinin A 20 KIU (Sigma-Aldrich, St. Louis, MO, USA). Then, it was homogenized, centrifuged at 3,000 g for 10 min and the supernatants collected for cytokine assay. Plates were coated with purified monoclonal antibodies reactive with cytokines IL-6, TNF-α, TGF-β (active form), IFN-γ, IL-4, IL-17, and IL-10 (BD Bioscience, San Jose, CA, USA), overnight at 4°C. In the following day, wells were washed, supernatants were added, and plates were incubated overnight at 4°C. On the third day, biotinylated monoclonal antibodies against cytokines (BD Bioscience, San Jose, CA, USA) were added and plates were incubated for 2 h at room temperature. Color reaction was developed at room temperature with 100 μl/well of orthophenylenediamine 1 mg/ml (Sigma-Aldrich, St. Louis, MO, USA), 0.04% H₂O₂ substrate in sodium citrate buffer. Reaction was interrupted by the addition of 20 μl/well of 2 N H₂SO₄. Absorbance was measured at 492 nm by ELISA reader (Bio-Rad, Hercules, CA, USA).

Sources of the compounds used in the study are given in parentheses, next to compounds name. Fluphenazine (Sigma, F0280000); Fluphenazine mustard (Enzo, BML-CA325-0050); Trifluoperazine (Sigma, 1686003); DT-061 (MedChem Express, HY-112929); Ophiobolin A (Santa Cruz, sc-202266); Calmidazolium (Santa Cruz, sc-201494); FTI-277 (BioVision, 2874); AMG-510 (MedChem Express, HY-114277); Trametinib (Bio-connect, SC-364639); Benzethonium chloride (Sigma-Aldrich, 53751). DMSO was from PanReac-AppliChem (cat. no. A3672, ITW Reagents). Caspase-Glo 3/7 assay kit was from Promega (G8090).

For the quantification of cytokines, the ileum were weighed and homogenized in PBS containing 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA), phenylmethylsulfonyl fluoride 0.1 mM (Sigma- Aldrich, St. Louis, MO, USA), benzethonium chloride 0.1 mM (Sigma-Aldrich, St. Louis, MO, USA), EDTA 10 mM (Synth, São Paulo, São Paulo, Brazil), and aprotinin A 20 KIU (Sigma-Aldrich, St. Louis, MO, USA). Afterwards, this material was homogenized, centrifuged at 3,000 g for 10 min and the supernatants collected for cytokine assay. Plates were coated with purified monoclonal antibodies reactive with cytokines IL- 10, IL-12 p70 and IL-1β/IL-1F2 (R&D Systems, Inc, USA), overnight at 4°C. Then, plate wells were washed, supernatants were added, and plates were again incubated overnight at 4°C. On the third day, biotinylated monoclonal antibodies against cytokines (R&D Systems, Inc, USA) were added on the plates and incubated for 2 h, at room temperature. Colour was developed at room temperature with 100 μl/well of orthophenylenediamine (1 mg/ml) and 0.04% (v/v) H₂O₂ substrate in sodium citrate buffer. The reaction was stopped by the addition of 20 μl/well of 2N H₂SO₄. The absorbance was measured at 492 nm using a Microplate Reader Model 680 (BIO-RAD).

QACs: benzalkonium chloride (BC), benzethonium chloride (BZ), cetylpyridinium chloride (CP), cetyltrimethylpyridinium chloride (CTAB), and domiphen bromide (DB) were purchased from Sigma-Aldrich (Steinheim, Germany).
The ILs used in this study were either (a) provided by Proionic GmbH (Grambach, Austria) with a nominal purity of >98%, (b) purchased from Iolitec (Ionic Liquid Technologies GmbH, Heilbronn, Germany) with a nominal purity of >98%, or (c) synthesized in our laboratory, according to the CBILS© route (CBILS is a registered trademark of Proionic GmbH; Kalb et al., 2005 , 2016 (link)). The following ILs were investigated as: ILs with one elongated alkyl side chain 1-decyl-3-methylimidazolium chloride ([C₁₀mim][Cl];a), 1-dodecyl-3-methylimidazolium chloride ([C₁₂mim][Cl];b), 1-methyl-3-tetradecylimidazolium chloride ([C₁₄mim][Cl];b), 1-hexadecyl-3-methylimidazolium chloride ([C₁₆mim][Cl];b) trimethyldecylammonium chloride ([TMC₁₀A][Cl];c), trimethylhexadecylammonium chloride ([TMC₁₆A][Cl];c). ILs with two elongated alkyl side chains dioctyldimethylammonium chloride ([DC₈DMA][Cl];c) and 1-3-didecyl-2-imidazolium chloride ([C₁₀C₁₀im][Cl];b). ILs with three elongated alkyl side chains trioctylmethylammonium chloride ([TC₈MA][Cl];a) and trioctylmethylphosphonium chloride ([TMC₁₀P][Cl];c).