Whole Blood Cytogenetic Analysis Protocol

Whole blood samples from each dose point were cultured in PB-MAX™ Karyotyping media (Life Technologies, Grand Island, NY), and incubated at 37 °C, 5% CO₂, 98% humidity. After 44 h, the media was refreshed with PB-MAX™ media supplemented with cytochalasin B (Sigma-Aldrich LLC, St. Louis, MO) at a final concentration of 6 μg/mL to block cytokinesis. This protocol, which uses small volumes of blood in multi-well plate format, is based on our earlier published protocol^{20 (link),26 (link)}. After a total incubation period of 72 h, the cells were harvested. The cells were treated with 0.075 M KCl solution (Sigma-Aldrich, St. Louis, MO) at room temperature for 10 min. After hypotonic treatment, the cells were fixed with fixative (4:1 methanol:glacial acetic acid). The fixed cell samples were stored at 4 °C (at least overnight), dropped on slides, allowed to air dry for 10 min and then stained with Vectashield^® mounting media containing DAPI (Vector Laboratories, Burlingame, CA). The slides were left overnight at 4 °C prior to imaging.