**4.1 General procedure for the cytokinesis-block micronucleus (CBMN) assay in lymphocytes**

The experimental procedure begins by extracting whole venous blood in heparinized tubes. According to the designed experiment, cultures are prepared with 6.3 mL

#### **Figure 1.**

*Human lymphocyte binucleated cells: a) Binucleated normal cell (BNC), b) Binucleated cell with micronucleus (MNi), c) Binucleated cell with nucleoplasmic bridge (NPBs), d) Binucleated cell with nuclear bud (NBUDs).*

of RPM1–1640 medium supplemented with non-essential amino acids, 0.2 mL of phytohemagglutinin, and 0.5 mL of whole venous blood incubated for 44 hours at 37°C. After this time, between 3 and 6 μg/mL of cytochalasin-B is added to avoid the division of the cytoplasm (blocking cytokinesis), and incubation is resumed until 72 hours are completed (**Figure 2**).

Once the 72-hour incubation period is finished, cells must be fixed with Clarke's solution and washed 3 to 5 times until a clear cell button is obtained. If necessary,

**Figure 2.** *The general procedure of the cytokinesis-block micronucleus assay in lymphocytes.*

impurities are removed with a trypsin solution. The cell button is transferred on slides and stained with eosin and methylene blue to record nuclear abnormalities (**Figure 1**) in a total of 1000 binucleated cells and count in 500 cells those mononucleated, binucleated, trinucleated, and tetranucleated cells to calculate the cell proliferation index (NDI) and also record the number of cells in necrosis and apoptosis as indicated in the protocol [45, 46].
