A total of 1×105 cells/ml cells were inoculated in a 35-mm-diameter petri dish
containing a glass cover slip, cultured for 1 day and synchronized with medium containing
0.4% FBS for 3 days so that the majority of cells were in G0 phase.
Subsequently, 1.0 mg/ml 5-bromo-2’-deoxyuridine (BrdU) reagent (BD Pharmingen, BD
Biosciences, USA) was added, and the cells were cultured in complete medium at 37˚C for 2
hours. Next, the culture solution was removed; besides, the cover slips were washed in
phosphate-buffered saline (PBS) three times; and the cells were accordingly fixed with
methanol for 10 minutes. The dried slides were subsequently blocked with 5% normal rabbit
serum (Beyotime, China), and nucleic acids were denatured with formamide (Sigma-Aldrich,
China). Subsequently, the cover slips were rinsed with PBS and then incubated with the
primary antibody anti-BrdU (Beyotime, China, 1: 500) at room temperature for 1 hour, while
the control group was incubated with PBS. Next, the nuclei of the cells were stained with
DAPI staining solution (Beyotime, China) for 2 hours at room temperature, and finally, the
number of BrdU-positive cells in 10 visual fields were observed and counted under a
fluorescence microscope (Olympus, Japan), and the average number of the BrdU positive
cells was calculated.
containing a glass cover slip, cultured for 1 day and synchronized with medium containing
0.4% FBS for 3 days so that the majority of cells were in G0 phase.
Subsequently, 1.0 mg/ml 5-bromo-2’-deoxyuridine (BrdU) reagent (BD Pharmingen, BD
Biosciences, USA) was added, and the cells were cultured in complete medium at 37˚C for 2
hours. Next, the culture solution was removed; besides, the cover slips were washed in
phosphate-buffered saline (PBS) three times; and the cells were accordingly fixed with
methanol for 10 minutes. The dried slides were subsequently blocked with 5% normal rabbit
serum (Beyotime, China), and nucleic acids were denatured with formamide (Sigma-Aldrich,
China). Subsequently, the cover slips were rinsed with PBS and then incubated with the
primary antibody anti-BrdU (Beyotime, China, 1: 500) at room temperature for 1 hour, while
the control group was incubated with PBS. Next, the nuclei of the cells were stained with
DAPI staining solution (Beyotime, China) for 2 hours at room temperature, and finally, the
number of BrdU-positive cells in 10 visual fields were observed and counted under a
fluorescence microscope (Olympus, Japan), and the average number of the BrdU positive
cells was calculated.