Coomassie brilliant blue

The proteinase activities of MMP-9 and MMP-2 were measured by gelatin zymography. 10% polyacrylamide gels copolymerized with gelatin (0.1%, G8150, Sigma) were prepared. H9c2 (5 × 10⁴ cells/mL) and HepG-2 (5 × 10⁵ cells/mL) cells were seeded at volumes of 0.5 mL in 24 well plates. After being pretreated with PNG extracts/fractions for 24 h, the cells were stimulated by recombinant rat TNF-α (10 ng/mL) for another 72 h. The supernatants were collected and loaded into each well of the same volume. Following 2 h of electrophoresis, the gels were washed with 2.5% Triton X-100 for 1 h at room temperature to remove sodium dodecyl sulfate (SDS). Gels were then incubated overnight at 37 °C in renaturing buffer (50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl₂, pH 7.5). After incubation, the gels were stained with 0.05% Coomassie Brilliant Blue (G-250, Sigma) in a mixture of methanol: acetic acid: water (5:1:4, v/v) and destained in the same mixture without Coomassie Brilliant Blue. Gelatinolytic activities were detected as transparent bands against the dark blue background. Zymograms were digitally scanned, and band intensities were quantified using GelQuantNET software V 1.7.8.

BAL fluid and BAL derived-EVs proteins were quantified using Pierce™ BCA Protein Assay Kit (Thermo Scientific Pierce Biotechnology, P.O. Box 117, Rockford, USA). A constant protein amount of (10 µg) of BAL-EVs and BAL was separated on 10% SDS-PAGE under reducing conditions (4×-loading buffer: 8% SDS, 40% (w/v) glycerol, 20% Beta-mercaptoethanol, 0.08% bromophenol blue) and transferred to a Hybond PVDF membrane (Amersham, GE, Chicago, IL USA) or stained with Coomassie brilliant blue (Merck, Kenilworth, NJ, USA). The membrane was then incubated with primary antibody directed against CD63 (AB0047-200, SICGEN at a dilution of 1:2500) and HRP-conjugated secondary antibody donkey anti-goat (Jackson ImmunoResearch, Cambridge, UK). Signals were detected by enhanced chemiluminescence reagents ECL Western blot Detection (Amersham, GE, USA). Intensity of the bands was quantified using ImageLab Software. HeLa total cell lysate was analyzed in parallel using the same conditions as a positive control for anti-CD63. Images obtained were processed by the open-source software ImageJ (imagej.net) for quantitation.