Cell migration assay

The effects of EL on migration potential of breast cancer MDA-MB-231cells were analyzed by cell migration assay (Justus et al., 2014). MDA-MB-231cells were seeded at a seeding density of 0.3 x 10⁶ cells per well in 6-well plates and incubated overnight at 37°C in 5% CO₂ incubator. An artificial wound was made with a sterile tip. Detached cells were removed by discarding existing media and replacing with fresh media containing different concentrations of EL; 25, 50, 75 μM and ethanol alone as negative control. The speed of wound closure and cell migration was quantified by taking snapshot pictures with an inverted microscope (Magnus-INVI, New Delhi, India) at 24, 48 and 72 hours time intervals. To determine statistical significance of the data, we carried out automated analysis of monolayer wound healing assay by analyzing the images in the computational tool; TScratch software, as described elsewhere (Gebäck et al., 2009).