Ultrasonic disintegrator

Aortic tissues and 20 μM montelukast-induced macrophages were homogenized using an ultrasonic disintegrator (Sonics & Materials, Inc., Newtown, CT, USA) in protein extraction buffer (CytoBuster; Novagen, Merck KGaA, Darmstadt, Germany) with 20 mM of ethylenediaminetetraacetic acid (Dojindo, Kumamoto, Japan) and 1 mM of phenylmethylsulfonyl fluoride (Thermo Fisher Scientific). Lysate protein concentration was measured using the Qubit® Protein Assay Kit and Qubit® 2.0 fluorometer (Thermo Fisher Scientific). An equal concentration of total protein was applied to each assay kit and detected according to the manufacturer's protocol for each ELISA kit (TIMP-1 and TGF-β1: R&D Systems, Minneapolis, MN, USA; TIMP-2: RayBiotech, Norcross, GA, USA; IGF-1: Mediagnost, Reutlingen, Germany; IL-1β, IL-6, TNF-α, and MCP-1: Bender MedSystems, Vienna, Austria; IL-10: Thermo Fisher, Waltham, MA, USA) [12 (link)]. Seven samples were analyzed per group.

The E. histolytica HK-9 strain was cultivated in Diamond’s medium as per previously published protocols (Diamond, Harlow & Cunnick, 1978 (link)). Briefly, the trophozoites, after 48–72 h culture, were chilled to dislodge the parasites and washed thrice with normal saline solution (NSS) by centrifuging at 120 g at room temperature (RT) for 5 min. The cell sediment was resuspended in an appropriate volume of NSS to give approximately 10⁷ cells/mL. The preparation was then sonicated with an ultrasonic disintegrator (Sonics & Materials, Newtown, CT) and centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was dialyzed against distilled water containing proteinase inhibitors (Roche Applied Science, Basel, Switzerland) and stored at −20 °C until further used as crude E. histolytica HK-9 antigen in the ELISA. The protein content of the antigen was determined using the standard method (Lowry et al., 1951 (link)).