Gfr matrigel matrix

Cancer multicellular spheroids were generated by using the hanging drop method [50 ]. Briefly, cells were detached with 2 mmol/L EDTA, counted, re-suspended in DMEM supplemented with methylcellulose (20%; Sigma) and GFR Matrigel matrix (1%, Corning) and incubated as droplets (25 μL) containing 10³ cells for 48 hours to generate multicellular aggregates. To generate “hybrid” cell spheroids, equal numbers of H1299-GFP cells and fibroblasts (transfected with either control or XRCC1 siRNA) were mixed (10³ cells per droplet). For “hybrid” spheroids invasion, either cancer spheroids alone or “hybrid” aggregates were plated on Matrigel and cultured for two days in presence of medium conditioned by fibroblasts. Tumour cell invasion were apparent as projections from the spheroids after two days of co-culture. The invasion was scored by counting the invasive protrusions around the spheroids. At least 40 aggregates were counted per condition.
For network formation assay, cancer cell spheroids were washed and plated on Matrigel. 4 × 10⁴ fibroblasts (transfected with either control or XRCC1 siRNA) were added to the spheroids as a single-cell suspension. Network formation and cancer cell migration were evident after 24 hours. Images were captured by using a Nikon 10X/0.30 Ph1 objective.

MDA‐MB‐231 cancer multicellular spheroids were generated by using the hanging drop method (Foty, 2011 (link)). Briefly, ZsGreen‐Control or ZsGreen‐RASSF1C transfected cells were detached with 2 mmol/l EDTA, counted, resuspended in DMEM supplemented with methylcellulose (20%, Sigma) and GFR Matrigel matrix (1%, Corning) and incubated as droplets (25 μl) containing 1,000 cells for 24 h to generate multicellular aggregates. Three‐dimensional aggregates were washed with medium, and either seeded and cultured on Matrigel matrix for 48 h or mixed with rat tail collagen (Serva; 2.0 mg/ml), 10× PBS, 1 M NaOH, and complete medium. Collagen gel‐spheroids solution was pipetted as a 100 µl drop‐matrix suspension into 12‐wells, polymerized at 37°C, replaced with medium, and cultured for another 24 h till single‐cell invasion from spheroids was apparent. Single‐cell morphology apparent as mesenchymal (elongated) projections from the ZsGreen transfected spheroids or as amoeboid (rounded) single cells invaded around from ZsGgreen‐RASSF1C transfected spheroids was imaged and captured by using Nikon 20×/0.8 Ph1 objective.

The treated hRMECs were inoculated into 24-well plates pre-coated with growth factor reduced (GFR) Matrigel Matrix (Corning Life Sciences). Following 16 h of culture, the capillary-like structures were photographed in five random fields under a microscope (Olympus) and measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell migration was evaluated by the wound-healing method. After 24 h transfection, cell monolayers were scraped, and images of the wounded area were captured at 0, 24, and 48 h by an inverted microscope (2× magnification). Images were analyzed using ImageJ software, and the change in the wound area over time was expressed as a percentage of wound closure. Pictures represent one of three independent experiments.
The Matrigel-based invasion assay was performed in Boyden chambers containing an 8 μm polycarbonate membrane coated with Matrigel (Corning Matrigel GFR Matrix, 2 mg/mL). Two days after transfection, 104 cells were added in suspension to the upper side of the Boyden chamber, while the bottom well contained 10% serum in the culture medium. After 24 h, migrated cells were fixed and stained with the DiffQuik Stain Kit (Baxter Diagnostics), quantified by analyzing different microscopic fields (10× magnification), and expressed as mean numbers of migrated cells. Data represent three independent experiments.
Two days after seeding, cell proliferation was assessed using the Resazurin Assay Kit (Immunological Sciences, Rome, Italy) as previously described [41 (link)]. The relative cell number was calculated from standard curves of resazurin fluorescence. Data represent three independent experiments, performed in triplicate.