Using flow cytometry, apoptotic/ necrotic cells were
detected by an Annexin V- FITC and propidium iodide (PI)
apoptosis assessment kit according to the manufacturer’s
protocol. In this assay, the cells, which were treated with
5-aza (5 μM), different doses of Alimta (3 and 12 μM)
alone and combined with 5-aza, were trypsinized, washed
with PBS and suspended in 100 μl 1x buffer. The cells
were then stained with AnnexinV/PI, incubated for 15
min in the dark at room temperature and analyzed using
a FACScan flow cytometer (Becton Dickinson, San, CA,
USA). The percentage of apoptotic cells was determined
by flow max software.
In order to evaluate cell cycle distribution, the control and drug treated cells
(106 /ml), as described above, were collected, fixed in 70% ice-cold Ethanol
and stored at 4°C for 2 hours. Then, cells were stained with DNA staining solution (PBS
containing 20 μg/ml RNase A and 20 μg/ ml PI) by incubating at 37°C for 30 minutes in the
dark. The cell cycle distribution was analyzed using a FACScan flow cytometry and data
analyzed with FlowJo software.
detected by an Annexin V- FITC and propidium iodide (PI)
apoptosis assessment kit according to the manufacturer’s
protocol. In this assay, the cells, which were treated with
5-aza (5 μM), different doses of Alimta (3 and 12 μM)
alone and combined with 5-aza, were trypsinized, washed
with PBS and suspended in 100 μl 1x buffer. The cells
were then stained with AnnexinV/PI, incubated for 15
min in the dark at room temperature and analyzed using
a FACScan flow cytometer (Becton Dickinson, San, CA,
USA). The percentage of apoptotic cells was determined
by flow max software.
In order to evaluate cell cycle distribution, the control and drug treated cells
(106 /ml), as described above, were collected, fixed in 70% ice-cold Ethanol
and stored at 4°C for 2 hours. Then, cells were stained with DNA staining solution (PBS
containing 20 μg/ml RNase A and 20 μg/ ml PI) by incubating at 37°C for 30 minutes in the
dark. The cell cycle distribution was analyzed using a FACScan flow cytometry and data
analyzed with FlowJo software.