I am performing an experiment to study cell cycle analysis after anticancer compound treatment. I am planning to use flow cytometry. Can someone suggest a protocol for this.
I just want to study number of cells in G1, G2, S and M phase.
For FCM: Collect the cells. The cells were washed three times with PBS, washed with 70% ice ethanol, centrifuges, discarded and washed three times with PBS. The cells were resuspended with 1ml of PI/Rigoberto X-100 (20ug PI/0.1% Triton X-100), staining solution containing 0.2 mg RNase A, 37*C staining15 min.
We have used DNA Prep from Beckman Coulter for many years, it works very well, you do not need to add RNAse (because propidium iodide binds both to DNA and RNA) since the kit has RNAse in it. It is a validated kit and very easy to use. Please note that to use an analysis program such as MultiCycle or ModFit is necessary for the calculations of the results.
@ Chu Qiao: Ok...How many cells should I plate for this. Is there a kit for PI/Rigoberto X-100 (20ug PI/0.1% Triton X-100).
@Gulderen: Can you name the kit please?
Hi Pratik,
Here, I attach my paper and a online protocol as a reference for staining your cells.
However, to answer your question about number of cells to seed, depends on the duration of your drug treatment.
You can write to me directly for further questions you may have.
Good luck..!
http://www.abcam.com/protocols/flow-cytometric-analysis-of-cell-cycle-with-propidium-iodide-dna-staining
Article N-nitroso-N-ethylurea activates DNA damage surveillance path...
@ Satish Bodakuntla: Hi..thanks..
I want some other details as well.
1. How many cells per well. Does it depend on number cells like 300,000 or more?
2. How do you synchronize all cells together. I read the reference paper. But it would be more clear if you can explain this.
"1. How many cells per well. Does it depend on number cells like 300,000 or more?"
I am not sure if I understood your question completely. As for the number of cells, it depends many factors including duration of the drug treatment.
For example, if you want to treat your cells for 6 hours, can seed 1 million cells per well (6 well plate). After 16-20 hours of seeding you can add your drug for 6 hours. After the drug treatment, you can proceed for FACS analysis.
However, as you are interested in cell cycle. I would suggest you to seed 0.5 million cells per well (6 well plate). Next day, add the drug for 24 hours (you should determine the IC50 for the drug at this time point and then use the optimal concentration of the drug for your assays). After drug treatment you can proceed for the FACS analysis.
" How do you synchronize all cells together. I read the reference paper. But it would be more clear if you can explain this."
Please find the following attachment.
Hope it helps.
Good luck..!
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