N diacetyl n methylcolchicine

In order to furnish successive visualization of sister chromatid exchanges (SCEs), a 5-bromo-2-deoxyuridine (Sigma-Aldrich) solution was added to culture initiation. Demecolcine (N-diacetyl-N-methylcolchicine, Sigma-Aldrich) was added to the cultures 70 h and 30 min after the beginning of incubation. The cell suspension was treated with hypotonic solution (0.075 M, KCl), three repetitive cycles of fixation in MeOH/CH3CO2H solution (3 : 1, v/v), centrifugation, and resuspension. Then, this cell suspension was dropped onto chilled, grease-free microscopic slides, air-dried, aged for three days, and differentially stained for the inspection of SCE rate according to fluorescence plus Giemsa (FPG) procedure. Well-spread thirty-second division metaphases containing 42–46 chromosomes per cell were scored for each treatment condition. Obtained values were estimated as SCEs per cell.

To visualize the SCEs, 5-bromo-2-deoxyuridine (Sigma Aldrich ^®) was added to the cultures at the beginning of incubation. Demecolcine (N-Diacetyl-N-methylcolchicine, Sigma Aldrich ^®) was added to the cultures after 70 h and 35 min of culture initiation. After completing 72 h, the cells were collected in a tube by centrifugation and treated with KCl (0.075 M) as a hypotonic solution. The cells were then fixed three times in cold methanol/acetic acid solution (3:1, v/v). The cell suspension was dropped onto cold slides and kept overnight. Air-dried slides were stained according to the Fluorescence Plus Giemsa (FPG) technique. For the scoring of SCEs, well-spread 25 metaphases containing 42 to 46 chromosomes per cell were counted, and the values were expressed as SCEs per cell.