Microscope slides were prepared after a standard 24- to 48-hour cell culture and harvested by using a 0.075M KCl hypotonic solution for 20 minutes at 37[degrees]C without
colcemid followed by 3 changes, at room temperature, of freshly prepared 3:1 methanolglacial acetic acid fixative.
Cell cycling was arrested in metaphase by addition of 10 [micro]g/ml
colcemid and incubating for 2-4 hours (Thermo Fisher Scientific[R], Paisley, UK).
Colcemid (Thermo Fisher, MA, USA), a cell cycle arresting agent, was used as a positive control at a concentration of 100 ng/ml.
Seis horas antes de la cosecha los medios fueron tratados con un pulso de BrdU y 1 hora antes con
Colcemid. Transcurrido este tiempo se cosecho cada medio.
For this purpose, the cells were incubated for 2h and 30 min in the vicinity
colcemid. KCL (0.075 M) was used as a hypotonic solution.
We have previously shown that oxidants such as hydrogen peroxide, menadione, and other chemicals including
colcemid, amoxicillin, and flavonoids of propolis can inhibit gap-filling during the repair of UVC-induced DNA lesions [3].
Then, metaphases are harvested by adding
colcemid (10 mg/l) for 120 min, followed by hypotonic KCl (0.075 M) treatment for 30 min, and fixation using stand 3:1 methanol-acetic acid.