4.7. Cytokinesis-Block Micronucleus (CBMN) Cytome Assay

Cells, 1 × 10⁵ MG-63 cells/well or 2 × 10⁵ U2OS cells/well, were seeded on cover glasses in 6-well plates containing complete DMEM and left to adhere overnight. After exposure to DiOHF, the exposure medium was replaced with DMEM containing 3 µg/mL cytochalasin B and cells were incubated for 40 h. The CBMN assay was a modification of Fenech’s protocol [58], conducted according to the OECD guideline 487 (In Vitro Mammalian Cell Micronucleus Test, 2016). After exposure, cells on cover glasses were washed with PBS, methanol-fixed at −20 °C for 10 min, dried, and stained with acridine orange. The samples were observed under an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan), equipped with a B-2A filter. Cytokinesis-block proliferation index (CBPI) values were calculated according to the OECD guideline 487 (2016) (https://www.oecd.org/chemicalsafety/test-no-487-in-vitro-mammalian-cell-micronucleus-test-9789264264861-en.htm (accessed on 14 January 2020)) for cytochalasin B treated cells.