Cellular proliferation was assessed using Sulphorhodamine B (SRB) and colony forming assays as described in Miko et al. and Fodor et al. [16 (link),97 (link)]. Cells were seeded in 96-well plates (4T1, 1500 cells/well; MDA-MB-231, 3000 cells/well; SKBR-3, 5000 cells/well; MCF7, 4000 cells/well; ZR75-1, 3000 cells/well; human fibroblast, 7500 cells/well) in complete medium and were cultured with different concentrations of IS for 24 h. Then, cells were fixed by the addition of 50% trichloroacetic acid (TCA, final concentration: 10%) and the plates were incubated for 1 h at 4 °C. Plates were washed five times in water and stained with 0.4% (w/v) SRB solution in 1% acetic acid. Unbound dye was removed by washing five times with 1% acetic acid. Bound stain was solubilized with 10 mM Tris base and the absorbance was measured on an automated plate reader (Thermo Labsystems Multiskan MS, Walthman, MA, USA) at 540 nm.
For colony forming assays, cells were seeded in six-well plates (4T1, 500 cells/well) and treated with the indicated concentrations of IS for seven days. After treatment, the plates were washed twice with PBS. Colonies were fixed in methanol for 15 min, dried, and stained with the solution of May-Grünwald-Giemsa for 20 min. Plates were washed with water and the colonies were counted using Image J software [98 (link)].
For colony forming assays, cells were seeded in six-well plates (4T1, 500 cells/well) and treated with the indicated concentrations of IS for seven days. After treatment, the plates were washed twice with PBS. Colonies were fixed in methanol for 15 min, dried, and stained with the solution of May-Grünwald-Giemsa for 20 min. Plates were washed with water and the colonies were counted using Image J software [98 (link)].
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