Hi Dan, You raise a good point here. When looking for Sub-G1 events you apply the same sort of gating strategy that you would use when doing regular DNA cell cycle analysis ie using area v width to define the singlet population. The way I normally do it is to use the first type of gate you show ie include all events that fall below the G1 peak and head towards the origin - I tend to exclude the events with very low area and width - a rule of thumb is to exclude all events below 10% of the G1 peak - this is a fairly arbitrary cut-off but if it is applied consistently will at least allow you to compare between samples. One thing that you have to be careful about is calling the events in the sub-G1 region 'apoptotic' events - all we can say is that these events have less DNA than a normal cell, we don't know why they do - they may have undergone specific DNA degradation via an apoptotic pathway ot they may have arrived there through some other pathway of oncosis/necrosis. As others have said, apoptosis is best studied not only with cytometric methods that look at different parts of the apoptotic pathway but at different time points following apoptotic induction. In our laboratory, we routinely look at membrane changes with Annexin, mitochondrial membrane potential changes with CMXRos, caspase activity and changes in cellular DNA. More information about how we look for apoptosis on the flow cytometer may be found by following the apoptosis link on my website. Incidently, the smear that you see is also often found in cell cultures that are contaminated either bacterially or by mycoplasma. A quick look down a fluorescence microscope can be instructive! Derek Dan Rosson wrote: > I'd like to ask my more learned flowlosopher colleagues a question on > gating for the purpose of quantitating sub G1 content for apoptosis > studies. I'm hoping Powerpoint illustrations can be attached to these > mass mailings. We all know that we should gate out doublets and > triplets in order to restrict a cell cycle analysis to single cells and > one of the ways of doing this is to plot Area vs Width in order to draw > a region. The Powerpoint attachment is a dot plot of such an experiment. > Notice the long trail of sub G1 events that are big, fat and wide. > Should these events be gated out as in the first histogram or left in as > in the second histogram? It seems to me that if they are two or more sub > G1 events are stuck together they're still apoptotic and should be left > in the gate otherwise it's cheating the apoptotic population of its due > representation. However, I've never seen a discussion of this. > So, if some of you have any insight into this, I'd like to hear it. ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Cancer Research UK, e_mail: derek.davies@cancer.org.uk London, UK mobile: 07790 604112 Web Page: http://science.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux *************************************************************************
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