Cell cycle analysis - Include floating cells or not?? (Mar/21/2006 )
Hi guys,
I am about to do a cell cycle analysis of cells treated with a drug that causes a lot of cell death. The procdure i was told to follow is:
1. Pull off media from cells.
2. Wash with PBS twice.
3. Add 1.5 ml of citrate buffer to the plates and put it away at -80 till ready to use.
4. Pull off the buffer (the cells come off), centrifuge add sol A and incubate at RT for 20 mins
5. Add PI. then assay using flow cytometer.
My question is: When i pull off the media i am going to lose cells that have already undergone apoptosis. will this have an effect? (according to me ..yes, I will lose cells in the subG0 phase). What do you guys think??
Should i include the floating cells or not??
thanks
p..
Anyone???
You're correct in saying that you'll lose cells that are well underway/have already undergone apoptosis by removing the floaters. I've always thought of floaters as late-stage cells, and depending on your assay you may want to keep that subpopulation in your experiment. For PI, I would keep them, as well as for basically any assay related to the measurement of apoptosis. i've also spoken with people who don't keep them and their experiments seem to work fine too, so in the end the floaters may actually contribute little to the measurement you are trying to make.
Elias, Thanks a lot for the reply..
Now at one of my concentrations my cells undergo massive apoptosis. So my interest in keeping the floaters is because they may help with the cell numbers. I dont think i can reliably use flow cytometry with low number of cells.
Have another question?? how do u harvest ur cells??
So just out of interest:
Isn't the occurence of cell death something different then how cells go through the cell cycle?
When you add so much of the drug that cells die, doesn't the question become about the way that the cells die (ie through apoptosis or not). Shouldend you be able to see if and where the drug is acting in the cell cycle (and possibly where you loose the cells) using a range of lower concentrations or (a range of) earlier time points.
Isn't the occurence of cell death something different then how cells go through the cell cycle?
When you add so much of the drug that cells die, doesn't the question become about the way that the cells die (ie through apoptosis or not). Shouldend you be able to see if and where the drug is acting in the cell cycle (and possibly where you loose the cells) using a range of lower concentrations or (a range of) earlier time points.
doing that also....
Sounds like a very potent drug, maybe a bit spooky....
good luck
Isn't the occurence of cell death something different then how cells go through the cell cycle?
When you add so much of the drug that cells die, doesn't the question become about the way that the cells die (ie through apoptosis or not). Shouldend you be able to see if and where the drug is acting in the cell cycle (and possibly where you loose the cells) using a range of lower concentrations or (a range of) earlier time points.
I believe you might be referencing the use of PI in studying cell death - in which case it is a commonly used approach to get a general number of dead/dying cells. Since PI measures absolute DNA content to place poulations within a point of the cell cycle, and dying cells have less DNA within them (for various reasons), one can measure sub-G1 DNA content to get an idea of the proportion of dead/dying cells in their experiment. Assigning some meaning to the measurement though would definitely require thinking along the lines of what you outlined above.