Hi Anne-Elen, Your plots look reasonable to me. Using samples with annexin and Pi alone can be useful for setting voltages but your best control is an untreated samples stained with both dyes which you can then compare with a treated sample. With analysis of these files, one important thing is consistency of gating particularly scatter gating - you really want to include all events that look like 'whole cells' and probably exclude debris which may be PI+ and may skew your percentages especially if there is a lot of cell death. The pattern of staining you are seeing isn't uncommon - there will be a spread of data especially of the annexin+ events. I am guessing that these are adherent cells of some sort - sometimes in these it can be hard to delineate where negative ends and positive begins. Quadrant markers don't always apply - I quite often use elliptical or polygonal gates - however you have to think about the biology, cells don't suddenly become apoptotic once they cross the boundary of a marker. Depending on your experiment it is also sometimes useful to consider the percentage of cells that are double negative to be the most accurate because you can at least define these more precisely. You should be consistent though where you put the markers once you have made sure that variation isn't due to cell number differences etc. As long as you are consistent with this you can compare between samples and/or treatments. Derek On 3/12/07 8:04 pm, "Anne-Elen.Kernaleguen@icm-mhi.org" <Anne-Elen.Kernaleguen@icm-mhi.org> wrote: > > Hello, > > I have some questions regarding Annexin V/PI gating. > > We are measuring CRP induced apoptosis on our cells using an annexin V/PI > staining. Our problem is that the cell populations are not always well > separated and sometimes ³shift² Š > > Usually, we are placing our negative/positive gate (the ³C² gate) using the > Annexin and PI single-stained cells (and itıs often easier to see the > positive/negative separation on histogram than on plots). But for some > patients (like patient 2), populations seem to shift toward the right side > of the plot. > Iıve put the plots of two staining on the PPT presentation : ³patient 1² is > what we usually observe, ³patient 2² is one of the problematic ones Š > > So here are my questions : > > 1- In the ³patient 1² case, do you think the gate is correctly placed ? (We > also tried to consider only the unstained sample, but since the Annexin > negative population seemed to shift to the right, we thought it would be > more accurate to consider the annexin stained sample : is that correct ?) > > 2- In the ³patient 2² case, the population seem to shift to the right for > the CRP sample. Could this be due to a problem in the tube (but itıs not > the only patient for which we observe this shift) ? Or is it normal ? > Therefore, do we have to consider the ³C² gate for all the samples ? Or > should we consider another one (like the ³G² one) for the CRP sample only ? > > Thanks for any help ! > > Anne-Elen Kernaleguen > Research Assistant > Montreal Heart Institute > Montreal, (Qc) Canada > > (See attached file: Apoptosis.ppt) > This attachment - 'Apoptosis.ppt' - 6.60 MBytes - can be viewed at > http://www.cyto.purdue.edu/MD-parts/86851424600ea123bbab3fbc1b8f45375f223cfe.p > pt > -- *************************************************************** Derek Davies, FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincolns Inn Fields, London, UK. Tel: (44) 20 7269 3394 FAX: (44) 20 7269 3479 mobile: 07790 604112 e_mail: derek.davies@cancer.org.uk Web Page: http://science.cancerresearchuk.org/sci/facs/ In tenebris lux ***************************************************************Received on Sat Dec 8 14:58:00 2007
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