I want to analyze cell cycle (apoptosis specifically) using FACS with propidium iodide staining. After collecting cell sample (control as well as treated), i fix it with 70% ethanol overnight. But i am afraid that cell may die during this interval, giving false results then. So i am asking if some of you has a suitable protocol, free of all problems. And if someone kindly tell me how you prepare Propidium iodide solution.
Well there is a very easy way with PI staining. You dont have to fix the cells and of course cells will die when you fix them. Just trypsinize or use PBS+EDTA solution to detach your cells, spin them, wash them two-three times with sterile PBS and your samples are ready for PI staining. Depending on the concentration of your PI you just need to add it to your samples before analyzing on FACS. I would also suggest that do not leave them very long in PI as PI is also toxic and cells will die if left long.
According to Léonce et al. (Léonce et al., 2001). To collect the cells, wash with warm PBS and use trypsin to detach the cells. When the cells were completely detached, add media and transfer the cell suspension to microtube. After centrifugation at 3,000 rpm for 5 minutes (depend on the cell, I used HepG2), the supernatant was removed using a pipette. The cell pellets were re-suspended in warm PBS. Then add cold absolute ethanol to each tube (the final conc of EtOH is 70%), mix with a pipette and a vortex mixer, and incubate at 4 oC for 1 hour to fix the cells. To remove ethanol, centrifuge at 4,500 rpm for 10 minutes and then renove supernatant. Wash the cells by re-suspension in 500 µl of warm PBS, centrifugation at 4,500 rpm for 10 minutes, and discarding of the supernatant. Finally, in the darkness, stain the cells with 0.5 mL of warm PI solution; 7 ml of PI solution consists of 0.35 ml of PI stock solution (1 mg/ml), 0.7 ml RNase A stock solution (1 mg/ml), and 6 ml of PBS, and incubate in darkness for 30 minutes. The samples were kept on ice until flow cytometric analysis.
Hope this help! I used this method and the result was good.
I perform apoptotic assays using Annexin V-APC and PI. This protocol uses live cells that aren't fixed, allow ingfor measuring early, late and necrotic cell death. I use PI at the concentration of 120ng/ml.
For cell cycle analysis even you can store sample at -20C for several days. you may keep the cells overnight as u mentioned above. For analyzing cell cycle profile, it does not make sense that cells are alive or dead. In Cell cycle analysis DNA contents are detected which can be detected in both live and dead cells. But for apoptosis its better to use a seperate protocol using Annexin V and PI, KIT. GOOD LUCK
You can even fix the cells with 100% ethanol for one hour at 4C and then pellet them at a very low speed. after this suspend the cell pellet in PI-RNAse solution ( i use the solution from BD- bio sciences) and then acquire the cells via FACS after 20minutes of incubation. Also remember the ethanlo being using for cell fixation should be chilled and should be added dropwise on the cell pellet with continues light vortexing.
You can please follow the link below to obtain certain valuable informations:-
http://www.abcam.com/protocols/flow-cytometric-analysis-of-cell-cycle-with-propidium-iodide-dna-staining
For how long I can store the stock solutions of PI and RNAse and at which temperature?
Thank you!
Hello Nataliya Bukhanova!
PI Soln. in water: 2-8°C for 6 months.
RNase in water: -20°C for 2 years
Hi, Can we store cells at -80C after fixation for cell cycle analysis?
I stored cells fixed in PFA and then suspended in 70% EtOH at -20C for up to two weeks with no problems. I think you could do longer term storage at -80C.
Even if I seed 80,000 cells/well for cell cycle analysis using PI staining, the number of cells I can see during the analysis is less than 500 distributed to different phases of cell cycle. A kind of peak I am getting is also scattered one rather. My worry is that my cells are washed away during the process of preparation. Could this be a reason? Thanks!
Is cell fixation in 100% ethanol recommended while preparing cells for PI staining?
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