Phase Contrast Microscopy of cells with annotation

Three breast cell lines with different morphology and mobility were used in this workspace, invasive breast cancer cell line MDA-MB-231 in mesenchymal morphology; non-invasive breast cancer cell line MCF7 in epithelial morphology, and normal breast cell line MCF10A in epithelial morphology. MDA MB 231 and MCF7 cells were grown in DMEM medium containing 10% fetal bovine serum while MCF10A cells were in DMEM:F12 (1:1) containing 5% horse serum, 20 ng/ml epidermal growth factor, 500 ng/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 μg/ml insulin. 

The cells were grown in incubators containing 5% CO2 and humidity at 37◦C. In terms of viability, proliferation, and infection, they were monitored daily by inverted light microscopy and multiplied with trypsin, paying attention to the doubling times of each cell culture. Periodic stocking was applied to ensure the continuity of the cells. Cells were frozen in a solution containing FBS and DMSO. Storage was carried out at -80◦C and then -196◦C in a liquid nitrogen tank. Then, by utilizing the Fiji distribution of ImageJ [12,13] individual cell boundaries are marked for images taken for single-cell analysis, and cell layer boundaries are manually marked for image analysis.