Demecolchicine

DMEM and Ham’s F12 50:50 mixture and GlutaGRO (L-alanyl-L-glutamine solution) were purchaed from Mediatech Inc (Herndon, VA). Cosmic Calf Serum was purchased from Hyclone (Logan, UT). All plasticware was purchased from BD Biosciences (Franklin Lakes, NJ). Dulbecco’s phosphate buffered saline (PBS), Gurr’s buffer, penicillin/streptomycin, sodium pyruvate and trypsin/EDTA were purchased from Life Technologies (Grand Island, NY). Crystal violet, calcium chloride, demecolchicine, lead chromate (PbCrO₄) and potassium chloride were purchased from Sigma-Aldrich (St. Louis, MO). Acetic acid and methanol were purchased from J.T. Baker (Phillipsburg, NJ). Giemsa stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Titanium dioxide nanoparticles, Aeroxide TiO₂ P25, were purchased from Nippon Aerosil Co, LTD (Tokyo, Japan). Microscope slides and cytoseal 60 slide mounting medium were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Ethyl alcohol, Embed-812 kit, flat embedding mold, gluteraldehyde, osmium tetra oxide, sodium cacodylate, sodium maleate and uranyl acetate were purchased from EMS (Hatfield, PA).

Demecolchicine (Sigma-Aldrich), at a final concentration of 0.005 μg/mL, was added to the cell cultures overnight to arrest the cells in a mitotic state. After detaching the cells they were treated with a hypotonic, 0.56%, potassium chloride solution for ten minutes, centrifuged and resuspended in a fixation solution, acetic acid:methanol, 1:3. After another round of fixation cell suspension was spread out on wet, cold slides. The slides were flame dried to improve the spreading of the chromosomes. Conventional Q-banding technique was used according to the International System for Human Cytogenetic Nomenclature, ISCN 2009, and cytogenetic analysis was performed with a Cytovision Image analysis system (Applied Imaging, Sunderland, UK). 20 metaphases were analyzed from each culture. The cells of the second production of NK-92 serum-starved cells were analyzed as described above. 23 metaphases were analyzed of each culture.