T4885

The following reagents were obtained: ketamine (Dopalen injectable^®, Paulínia, Brazil), xylazine (Anasedan injectable^®, Paulínia, Brazil), Tween 80 (Synth^®, Diadema, SP, Brazil), hexadecyltrimethylammonium bromide (HTAB) (Sigma Aldrich^® H5882, Steinheim, Germany), o-dianisidine dihydrochloride (Sigma Aldrich^® D3252, Steinheim, Germany), dextran sulphate sodium salt (DSS)—colitis grade (36,000–50,000 MW, cod. 02160110-CF, MP Biomedicals, LLC, Solon, OH, USA), hydrogen peroxide (Synth^®), horseradish peroxidase (Sigma Aldrich^® P8250, Steinheim, Germany), trichloroacetic acid (TCA) (Sigma Aldrich^® T4885, Steinheim, Germany), reduced L-glutathione (Sigma Aldrich^® G-4251, Steinheim, Germany), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma Aldrich^® N-7505, Steinheim, Germany), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma Aldrich^® D-8130, Steinheim, Germany), and buffer RIPA (Sigma Aldrich^® R0278, Steinheim, Germany). IL-1β mouse, IL-10 mouse, CXCL-MIP-2 mouse, and TNF-α mouse were obtained from R&D Systems^®, Inc. (Minneapolis, MN, USA).

Purified OMVs [resuspended in HEPES (HEPES is 10 mM + 0.85% NaCl adjusted to pH7.4 and filter sterilized)] were thoroughly mixed with cold 100% trichloroacetic acid (TCA) stock solution (Sigma-Aldrich catalog #T4885) to make a final concentration of 20% TCA. Samples were incubated on ice for 30 min then centrifuged at 13,200 RPM (14,220 × g) for 30 min at 4°C. The supernatant was removed and the pellet was resuspended in 0.5 ml ice-cold acetone. The samples were centrifuged at 13,200 RPM (14,220 × g) for 15 min at 4°C. The supernatant was removed and each pellet was resuspended in HEPES and 4x RSB (Reducing Sample Buffer, Invitrogen catalog #NP0008) in a 3:1 ratio.