Matrigel membrane matrix

HMVP2 cells and spheroids were cultured for three days. Following trypsinization, cells were washed twice, re-suspended in a concentration of 2 × 10⁶ cells mixed with 200 μl 45% RPMI medium + 5% FBS + 50% matrigel membrane matrix (BD) and then the mixture was injected subcutaneously (SC) in a dorso-caudal location of two month old male FVB/N mice (N = 10). Mice were slightly sedated with isofluorane prior to injection. Similarly, approximately 200 spheroids resulting from an initial culture of 2 × 10⁵ cells were washed and re-suspended in fresh 45% RPMI medium + 5% FBS + 50% matrigel (200 μl total volume) and then injected subcutaneously (SC) in a second set of mice (N = 10). Mice were observed daily for tumor formation and small SC masses (~5 mm in diameter) were seen after two weeks of injection. Tumor masses were then measured twice a week with a digital caliper and tumor volume was calculated using the formula, tumor volume = ½ length × width² [60 (link)].

Invasion and migration assay were performed in triplicate using Transwell chambers coated with or without Matrigel membrane matrix (BD Biosciences, USA) as described in the manufacture’s protocol. For the invasion assay, cells were seeds onto a Matrigel-coated membrane matrix present in the insert of a 24-well culture plate 24 hours after transfection. The cells that did not invade through the pores were carefully wiped out with cotton wool. Then cells located on the lower surface of the chamber were stained with 0.1% crystal violet (Sigma) and counted. These experiments were repeated three times.