2 replies to this topic

[Elioelio]

Hi there,
I'm in my 3rd year of a PhD and am about to do loads of cell cycle analsis on my samples. I've been using PI only on ethanol fixed cells. However, I want to get the best results possible so I'm wondering if I should be using an additional stains.

Thing is, I'm pretty new to flow cytometry and have set up cell cycle analysis using PI. The guy overseeing the flow cytometers in my institute has left now, so I've noone really to help me out if I want to set the cytometer up with another stain/colour (sorry for the bad uderstanding - I am new to this!).

So, basically what I want to know is will PI staining give me a good result for cell cycle analysis? Or will I be wondering I'd get better results with a different stain?

I'm using DLD1 and HT29 cells (adherent) and simply want to know what phase of the cycle they're in.

Thanks for any help!

[pcrman]

PI staining is pretty standard assay for determining cell cycle distribution. Even for this simple and standard assay, proper setting of the machine and gating during data acquisition is very important. If you decide to learn the ropes and DIY, you need to play a lot with the machine. Prepare a lot of untreated cells of cell types and use the cells to get the basic settings right. The order of setting different parameters is also important. Attached you can find a file I compiled from the internet and find very useful.

 FACS PI cell cycle.pdf   121.5KB   1670 downloads

[Zee]

PI is a good standard for looking at cell cycle. However, if you are wanting to look at specific stages within the cell cycle, it is considered a crude assay. For example, if you want to look at apoptosis, Annexin V is typically used. If you want to look at proliferation, I would recommend performing an EdU staining (equivalent to a BrDU assay but a little simpler to do in lab). For both of these experiments, PI analysis is done in conjunction with the additional stainings, either for Annexin V or EdU, that tell you more about what the cells are doing. Most companies have kits and detailed protocols for both of these, including invitrogen and eBiosciences. (I usually recommend using invitrogen's SOPs while the reagents are cheaper from eBiosciences.)