Matrigel matrix

Single-cell suspensions at designated concentrations were combined with equal volume of Matrigel Matrix (R&D Systems, USA). Then the mixture was injected subcutaneously into each side of the hind leg of BALB/c nude mice. After tumor cell transplantation, the tumor dimensions were measured every other day and the volume was calculated using V = (length × width^2)/2. When the volume reached about 1 cm [3 (link)], the mice were sacrificed and the neoplasm was excised for weighting. All protocols were approved by the Institutional Animal Care and Use Committee of Southwest Medical University and conducted with humane animal care.

Transwell culture inserts (Costar, Cambridge, MA) were used for the assessment of cell migration and extracellular matrix invasion [8 (link)]. In migration assay, 1 × 105 cells in 200 μl of serum-free RPMI were seeded on top of transwell and incubated for 8 hours. For invasion assay, the filter was coated with a thin layer of Matrigel matrix (R&D System, Minneapolis, MN). The 1 × 105 cells were seeded onto the coated matrix and incubated for 18 hours. The number of migrated and invaded cells were counted at 200 × magnification under a light microscope. To determine anchorage independent colony formation assay, six-well plates were first layered with 1ml 0.7% low-melting point agarose in PBS. In the second layer, 100 cells per well were suspended in 1 ml RPMI containing 0.35% low-melting point agarose. 1ml RPMI was covered on the second layer. The plates were incubated for 4 weeks and then washed by PBS, fixed in 4% paraformaldehyde, and stained with 0.5% crystal violet. Colonies with a dimeter greater than 1mm were counted under an inverted microscope.