Azo dye impregnated collagen

Evaluation of the intracellular cysteine protease activity in total amebic lysates of treated trophozoites with E-64, ACh 1, 0.01, 0.0001, or 0.000001 µM as previously described (Bosch et al., 2012 (link); Dolabella et al., 2012 (link)). 100 µg of amebic extract and 2 mg of azo dye-impregnated collagen (Sigma-Aldrich, St Louis, Missouri, USA) resuspended in 500 µL of protease activation buffer (100 mM Tris pH 7.0 and 10 mM CaCl2), were incubated at 37°C for 18 h. After this reaction was stopped by adding 500 µL of 10% TCA. Then samples were centrifuged, collagen fibers were discarded, and supernatants collected for spectrophotometric determination at 540 nm.

Dimethyl sulfoxide (DMSO), 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT), collagenase, azo dye-impregnated collagen, and 2′, 7- dichlorodihydrofluorescein diacetate (DCFH2-DA) were purchased from Sigma Co. (St. Louis, MO, USA). Phosphate-buffered saline (PBS), penicillin/streptomycin (P/S), Dulbecco’s modified Eagle’s medium (DMEM), F-12 medium, and fetal bovine serum (FBS) were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). Antibodies against p-p38, p-JNK, p-ERK, GAPDH, p-c-Jun, nucleolin, NF-κB p65, and NF-κB p50 were purchased from Cell Signaling Technology (Beverly, MA, USA). The ELISA kits used for analysis of human MMPs, interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) were purchased from GE Healthcare Life Sciences (Exeter, Devon, UK). The PIP EIA kit was purchased from TaKaRa Bio Inc. (Kusatsu, Japan). All other chemicals used in this study were of analytical grade.

The ERM-certified PM material (ERM-CZ100 and PM₁₀-like); dimethyl sulfoxide (DMSO); azo dye-impregnated collagen; fluorescent probe 2′; 7′-dichlorodihydroflurescin diacetate (DCFH-DA); 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT); and MMP-1, 2, 8, 9, and 13 ELISA kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). The phosphate-buffered saline (PBS, 1X), Dulbecco’s modified Eagle medium (DMEM), Ham’s Nutrient Mixtures medium (F-12), fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). Antibodies against NF-κB p50 and p65; phosphorylated AP-1 (p-c-Jun); nucleolin; MAPKs including p-ERK, p-JNK, and p-p38; and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). PIP EIA kit was purchased from TaKaRa Bio Inc. (Shiga, Japan). All other chemicals used in this study were analytical grade.