Invadopodia Clause Samples

Invadopodia. Formation of invadopodia is considered indicative of invasive potential in vitro and in vivo (▇▇▇▇▇▇ et al., 2011; ▇▇▇▇▇ et al., 2014). We sought to examine the invasive potential of our cell lines knowing that MB-231 cells are widely used in invadopodia research (▇▇▇▇▇ et al., 2011) . In order to detect invadopodia formation in vitro, cells were seeded on glass coverslips previously coated with TRITC-conjugated gelatin. To allow invadopodia formation to occur cells were incubated at 37°C for 3h and were subsequently fixed with PFA and stained with Phalloidin 488 resulting in green stained cells over a red stained matrix (Hashim et al., 2013). Cells were considered able to make invadopodia when the actin puncta revealed by the phalloidin stain indicating the protrusive structure of invadopodia coincided with black holes in the red gelatin indicating matrix degradation (▇▇▇▇▇▇ et al., 2013). Out of the 4 cell lines that were subjected to the assay the HCC38 and MB-436 cells did not make any invadopodia (figure 3.7). Interestingly, reviewing the literature revealed that there is no published evidence of HCC38 or MB-436 cells being used in invadopodia assays. Together, these observations suggest that the HCC38 and MB-436 cells do not make invadopodia. As expected, the MB-231 cells exhibited definitive evidence of invadopodia formation and activity (figure 3.7A, 3.7B) which is consistent with their widespread use in invadopodia research. The BT-549 cells showed very little evidence of invadopodia presence at the 3h incubation as only a few very small invadopodia were detected. This observation suggested that the BT-549 cells might require longer incubations on the gelatin matrix to form active invadopodia. Further characterisation of the invadopodia capacity of the BT-549 cells was thus warranted. Invadopodia formation at 3 hours Figure 3.7 Invadopodia assay at 3h. (A) Cells were seeded on TRITC conjugated gelatin coated coverslips and incubated for 3h at 37°C. Coverslips were then fixed and stained with Phalloidin 488 (green) and cells were imaged. Scale bar corresponds to 10μm (B) Percentage of cells making invadopodia was determined for each individual experiment (N=3). Bars represent the mean percentage of cells making invadopodia and error bars represent the SEM.