Ecm gel from engelbreth holm swarm murine sarcoma

Skeletal muscle primary cells were isolated as previously described (Liu et al., 2015 (link)). Briefly, hind-limb muscles from 8 week old from 2 C57/Bl6J mice were collected and minced in wash media containing Ham’s F-10 supplemented with 10% fetal bovine serum (FBS) and 1% Streptomycin/Penicillin. Muscle tissues were then incubated in 800 U/ml Collagenase type II solution (Worthington, Cat. LS004176) at 37°C for 1 hr with gently agitation. After centrifugation, pellets were resuspended in wash media supplemented with 1000 U Collagenase type II and 11 U Dispase (Gibco, Cat. 17105–041), and incubated at 37°C for 30 min with gently agitation. Next, samples were triturated with a 20-gauge needle, centrifuged and resuspended in wash media. Cell suspensions were filtered using a 40 μm nylon cell strainer (Fisher Scientific, Cat. 22363547), centrifuged, and resuspended in Ham’s F-10 supplemented with 20% FBS, 1% Streptomycin/Penicillin, and 2.5 ng/ml basic fibroblast growth factor (bFGF, PeproTech, Cat. 100-18B-100UG). Cells were pre-plated for 30–40 min in an uncoated dish. Unattached cells were transferred to ECM-coated (ECM Gel from Engelbreth-Holm-Swarm murine sarcoma, Sigma, E1270) dishes. Plates were incubated at 37°C, 5% CO₂. Media was replaced until day three after plating, thereafter, media was replaced every 2–3 days.

The proteolytic activity of cell culture media was measured by zymography. In brief, 20 μL of serum-free conditioned media was loaded onto each well in 2× Laemmli loading buffer under non-reducing conditions (without β-mercaptoethanol and boiling) on 7.5% modified acrylamide gels containing 3 μg/mL casein (α-casein from bovine milk, Sigma Aldrich), 5 μL/mL fibronectin (Sigma Aldrich), and 10 μL/mL matrigel (#E1270, ECM Gel from Engelbreth-Holm-Swarm murine sarcoma, Sigma Aldrich). After electrophoresis at 200 V for 30 min, the gels were washed with 2.5% (v/v) Triton X-100 for 30 min and incubated for 18 h in a buffer solution containing TRIS (pH 7.5) and 10 mM CaCl₂ at 37 °C. After fixing in methanol–acetic acid–water (5:1:4) for 30 min, the gels were stained in 0.1% (w/v) Coomassie Brilliant Blue R-250 solution (dissolved in the fixation buffer) for 30 min. Areas of enzyme activity appeared as transparent bands on the blue background. The intensity of the bands was determined by densitometry using the free ImageJ (Version 1.50b, NIH, Bethesda, MD, USA) software.

Assays were performed using 6.5 mm Corning^® Transwell^® polycarbonate membrane cell culture inserts with 8 μm pore size (cat #3422; Sigma-Aldrich, St. Louis, MO), as previously described⁸¹ . The upper surface of the filter was coated with 40 μL of water-diluted extracellular matrix (ECM) gel from Engelbreth-Holm-Swarm murine sarcoma (final concentration 25 μg/mL; Sigma-Aldrich, St. Louis, MO), and left to dehydrate overnight, and rehydrated 2 hours prior to cell seeding with 50 μL of serum-free DMEM per transwell insert. Cells were grown to approximately 40% confluence under normal conditions, and transferred into reduced serum (2% FBS) medium for 32–34 hours prior to seeding. Cells were harvested, resuspended in serum-free DMEM, and 2.5 × 10⁵ cells in 100 μL was added to the upper chamber (total 150 μL of cell suspension per transwell, including 50 μL of rehydration medium added earlier). To the lower chamber, 600 μL of pharmacological treatment in DMEM supplemented with 10% serum (chemoattractant) was added, and cells were incubated for 24 hours at 37 °C in 5% CO₂. Non-migrated cells were scraped from the upper surface of the membrane with a cotton swab; migrated cells remaining on the bottom surface were counted after staining with crystal violet⁸² .