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.
The addition of colchicine (or colcemid
) pretreatment results in mitotic arrest and that treatment of arrested cells with a hypotonic solution like potassium chloride improved the yield and quality of metaphases spreads.
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).
(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 .
At 46th hour of incubation, the cells were blocked at metaphase by adding colcemid
at a final concentration of 0.1 [micro]g/mL and the cultures were further incubated until 48hrs.
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.