I am using propidium iodide for cell synchronization and a ki-67 analysis, and sometimes my peaks are not normal, I mean, instead of having two well defined peaks (for G1 and G2 populations), I have only one "fat" peak.
My cells are Hela and HEK293 and the protocol is the following:
- Tripsinization 3 minutes
- Add DMEM with FBS, centrifuge for 3 minutes at 300g
- Remove the supernatant and add 1 mL of 70% ice cold et-OH
- Fix the cells for 4 hours at 4ºC
- PI + RNAsa at 37ºC 1hour
I would be really gratefull if you could help me.
Thanks,
Abril (PhD Student).
Hi Abril,
Can I suggest you to avoid a cell fixation prior to stain with PI? I am not a specialist of KI-67 staining but I performed a lot of cell cycle analysis during my PhD and I found that sometimes it can help. You should also count your cells and prepare samples containing the same number of cells. I think you can reduce your incubation time with PI to 30 min. Are you sure there is no interference between PI and your secondary antibody for KI-67 detection ?
Guillaume
Dear Abril,
I recommend you to synchronize your cell by overnight serum starvation but be careful about HEK 293 cells especially because they are so fragile. (Maybe you can decrease the FBS level to 2 %). Then you start to collect the cells and perform your experiment.
On the other hand, the incubation time and amount of PI was also be the problem I previously use 40ug/ml PI and 30 min incubation.
I hope you will reach success soon.
Yunus
Hi Abril Sanchez..
I think you have to check your RNase concentration (it should be 5 ul from 10mg/ml stock per sample), insuffcient of RNase can casue problem like this and check your cell condtion.
Dear Guillaume, I don't know if I can avoid fixing the cells because I am doing the time course for 12 hours and I have to wait until the next day to do the facs, so maybe the cells dye and my population is not well defined.
Yes I am sure that there is not any interaction between the two fluorochromes.
Dear Yunus, I can not do a serum starvation because I need to set up this protocol to synchronize stem cells later :(
As far as the PI is concerned, I am using a 20 ug/mL concentration.
Dear Pasupathi, the RNAse concentration I am using is 100 ug/mL (so I put 5uL of RNAsa 10mg/mL to every 500 uL of sample.
In the case of ki-67 I am doing the PI-RNAsa incubation for 30 min (just as the protocol says), but there is not any improvement.
Dear Abril,
May I suggest to try this PI staining solution I developed ?I
I understand this is hard for you to avoid cell fixation because you probably can't run the analysis after your 12h time course, that's very long day. Anyway, after collecting your cells, you can directly apply my PI solution for 30 min then perform the analysis.
Alternatively, may I advise you to test my attached protocol ? It includes an overnight cell fixation that allows you to collect all your samples then run the analysis with all of them at once.
I hope it will be helpful.
Guillaume
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