Matrigeltm matrix

The abilities of cell migration and invasion of HepG2 cells were examined by using 24-well Transwell chambers with 8 μl pore size (BD Biosciences). Briefly, after transfection for 48 h, the cells (2 × 10⁵ cells/well in 200 μl serum-free culture medium) were cultured in the upper culture chamber, and the lower chamber was filled with 600 μl complete medium. For cell invasion assay, the upper culture chamber was covered with BD MatrigelTM Matrix (BD Biosciences). After incubation, the transwell chamber was removed, and the medium in each well was discarded. The cells were washed twice with PBS, and fixed with methanol for 30 min (Sigma-Aldrich, St Louis, MO, USA). After this, the non-traveled cells were gently removed from the upper surface of the filter with a wet cotton swab. Subsequently, the traveled cells were stained with 0.1% crystal violet (Merck, Darmstadt, Germany) for 20 min, and these cells were counted in five random fields under a microscope (Olympus, Tokyo, Japan; 40× magnification).

Mice were anesthetized with Pentobarbital (50 mg/kg, intraperitoneal; Narcoren^®, Boehringer, Ingelheim, Germany). After thoracotomy and preparation, the left anterior descending coronary artery (LAD) was ligated. After 45 min each mouse received an intramyocardial injection of MACS^® buffer (10 µL; Miltenyi Biotec) mixed with 10 µL of BD Matrigel^TM Matrix (BD Biosciences, San Jose, CA, USA). For cell application 100,000 (low dose, L) or 500,000 (high dose, H) stem cells of the respective source suspended in 10 µL of MACS buffer were mixed with 10 µL of BD Matrigel^TM Matrix. Injections of 4 × 5 µL were given along the border of blanched myocardium and ligation was removed. SHAM operated mice underwent identical surgical procedures without LAD-ligation but followed by intramyocardial MACS^® buffer/BD Matrigel^TM Matrix injection without cells.

The 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) colorimetric 18 (link), soft agar 16 (link)-20 (link) and matrigel (BD Matrigel^TM Matrix, BD Biosciences, Franklin Lakes, NJ) invasion 16 (link)-20 (link), 23 (link) assays for measuring the viability, growth, and invasion capability of tumor cells were conducted as previously described.

Briefly, the 8-well chamber slide (BD Bioscience, catalog number: 354108) is precoated with 80 µl growth factor reduced BD Matrigel^TM Matrix (BD Biosciences, catalog number: 354230) per well. Then the chamber slide is transferred to a cell culture incubator to allow matrigel solidification for at least 15 min. Five thousand cells for MCF-10A in assay medium containing 5 ng ml⁻¹ EGF and 2% Matrigel were seeded in each well. Medium were replenished every 3 days. Images of spheres with defined scales were subjected to the ImageJ computer program to determine the area covered by each sphere, and the diameter of that sphere was then calculated based on the circle formula.

Mice were anesthetized with 50mg/kg Pentobarbital (Vetmedica GmbH, Ingelheim, Germany) intraperitoneal injection. After thoracotomy and preparation, the left anterior descending coronary artery (LAD) was ligated. After 45 min each mouse received an intramyocardial cell injection. For cell treatment, 100,000 stem cells were suspended in 10 µL PBS containing 0.5% BSA and 2 mM EDTA and mixed with an equal amount of reduced growth-factor BD Matrigel^TM Matrix. The same experimental set-up was applied to control groups using cell suspension buffer and Matrigel^TM for application. Four injections, 5 µL each were given along the border of the blanched myocardium. Subsequently, the ligation was removed. Healthy control (SHAM)-operated mice underwent identical surgical procedures without left anterior descending coronary artery (LAD)-ligation.

Invasion assays were performed using modified Boyden chambers [33 (link)] coated with Matrigel. 8 μM pore transwells (Falcon HTS FluoroBlock Inserts, BD Biosciences, Bedford, MA, cat. # 351152) were pre-coated with 30 μL of BD Matrigel^TM Matrix (BD Biosciences, Bedford, MA, cat. # 354263) for 2 hours at 37°C. Glioma cells (50,000 cells) were placed on top of the membrane in the upper chamber in 150 μL of DMEM and microglia (50,000 cells) were placed in the lower chamber in 500 μL of DMEM. The control group did not contain microglia. Invasion was allowed to proceed for 18 hours at 37°C, 5% CO₂. In migration assays, Matrigel that mimics the extracellular matrix was not used. Migration assays were performed for 5 hours at 37°C, 5% CO₂.
In both assays, migratory cells were fixed with 100% methanol and stained with 40 μg/μL Propidium iodide (PI). Images of the cells that migrated to the lower chamber were captured using an inverted microscope Olympus BX51WI (Olympus, Shinjuku, Tokyo, Japan), Qcolor 3 camera (Olympus) and Q-capture Pro software (Olympus) and the number of migrating and invading cells on the underside of the filter were counted. The mean of the total invading or migrating cells was determined from 3 independent experiments.