Ethylenediamine tetraacetic acid 1 4na

TNBC MDA-MB-231 cells and human endothelial cell line (HUVECs) were seeded and transferred into a 12-well plates containing low serum medium (1 x 10⁴/well) for 1 day, 2 days and 3 days. Next the cells were washed twice with phosphate-buffered saline (PBS), pH 7.4, and trypsinized using trypsin-ethylenediamine tetraacetic acid containing 0.05% trypsin, 0.53 mmol/L ethylenediamine tetraacetic acid I 4Na (Gibco/Invitrogen, New York, NY). The resuspended cells were then subjected to a trypan blue dye exclusion assay and the positive cells counted using a hemocytometer.

MDA-MB-231 and MDA-MB-468 cells were transferred into low serum culture medium at a cell density of 1 ×10⁴ per well using 12-well plates. After 1, 2, 3 and 4 days of seeding, the cells were washed twice with phosphate buffered saline (PBS), pH 7.4, and trypsinized using 0.5 mL trypsin-ethylenediamine tetraacetic acid (0.05% trypsin, 0.53 mL ethylenediamine tetraacetic acid I 4Na, Gibco/Invitrogen, New York, NY). Next, the suspended cells were re-suspended in fresh culture medium, and this was followed by counting of cell numbers using a haemocytometer-based trypan blue dye exclusion cell quantification assay system. Cell cycles were calculated as cell cycle fraction percentages, namely, sub G₀/G₁ phase, G₀/G₁ phase, S phase, and G₂/M phase.