All, I am interested in determining the immunoreactivity fraction of antibodies by flow. I would appreciate any advice or input that any one has. Thank you . ____________________________________________ Jennifer L. Huntington Staff Biologist GE Global Research One Research Circle Niskayuna, NY 12309 T 518 387 7968 F 518 387 7765 huntingt@research.ge.com ____________________________________________ -----Original Message----- From: Derek Davies [mailto:derek.davies@cancer.org.uk] Sent: Thursday, March 30, 2006 3:35 PM To: cyto-inbox Subject: Re: Good apoptosis kits for flow cytometry, combining kits, & differentiating WBC populations OK lets try this one again, only a bit of the message made it through yesterday...... Hi Mark, It may be a simple question but I think it may have a complicated answer! As you are aware there are many ways of assessing apoptosis and cell death by cytometry (flow and static). The drawback of most of them is that they identify something that happens in apoptosis but one that always specific for apoptosis and which also may not occur in some pathways after induction. DNA fragmentation is normally a late stage manifestation of apoptosis (unless it is DNA damage that triggers the cell death) and many people will want to identify earlier changes. As you say annexin binding to externalised phosphatidylserine is often used. This has an advantage in that it is performed on unfixed cells all methods that can do this have the advantage that you can identify live cell, early apoptotic cells (annexin +ve but dead cell dye ve) and late apoptotic/dead cells (annexin +ve, dead cell dye +ve). However there are several apoptotic events that can be uptream of ps externalisation. What they are will likely depend on the method of apoptosis induction, the cell type and so on. We routinely look for changes in mitochondria using dyes such as TMRE (tetramethylrhodamine ethyl ester), the MitoTracker dyes, JC1 etc. In general these dyes pick up cells that are *probably* committed to apoptosis but arenıt annexin positive. It is also possible to look for caspase activation by using substrate assays or antibodies to activated sites. Mostly I donıt use kits for these but buy reagent separately but combination of assays is possible. One of the beauties of flow is that we can combine several of these methods as we are getting multiparametric information on a cell by cell basis. We find that we get the most information again when you can run unfixed cells so as too be able to positively discriminate dead cells. The dyes that you can combine will be restricted by the hardware you have available clearly a 3 channel cytometer wont be as versatile as a 12 channel one. To give you a couple of examples, we have looked at Annexin, DAPI and TMRE together (in a mixed GFP population); annexin, LDS751, Hoechst and TO-PRO-3; annexin, TMRE, caspase activation and DAPI and so on. As long as your emissions are separate you should be able to combine methods although a word of caution I find that controls for these are critical. One slight drawback of a live cell assay is that you have to remember you are seeing a snapshot of the population as it is measured to get some idea of apoptosis kinetics it is necessary to measure at different time points after apoptosis induction. Another thing to bear in mind is what exactly your cytometric method is measuring. This will be important when interpreting data for example cells that appear in the OEsubG1 regionı in a PI histogram following ethanol fixation wont be the same cells that appear in that region following aldehyde fixation and TUNEL staining. Also your suggestion of annexin binding and TUNEL may be technically possible but tricky on a flow cytometer (you would have to do the annexin binding then fix and do the TUNEL staining and you will lose the ability to distinguish cells that were dead prior to fixation unless you also add in ethidium monoazide or one of the new MolProbes dyes). However this could be done on a static cytometer by first staining for annexin then fixing and TUNEL staining and the merge the data files Professor Darzynkiewicz in NY has done a lot of elegant work on the Laser Scanning Cytometer like this. I may have to leave the wbc differentiation to an expert! Hope the rest helps though! Derek On 24/3/06 10:01 pm, "Hollier, Mark J" <etu4@CDC.GOV> wrote: > Hi all, > > I have a simple question (yes another question!). In the future I will be > wanting to analyze apoptosis by flow cytometry by methods other than the TUNEL > assay. To start with I want a method that will suggest overall apoptosis > levels compared to controls, using methods that do not rely on DNA > fragmentation. I would very much appreciate suggestions as to which kits are > the best, and reasons why. I was thinking that the externalization of > phosphatidylserine would be a good choice, would anyone agree? Or would > something like caspase activity be a better choice? Reasons for the proıs and > conıs of all suggestions would be very much appreciated. > > On another front, does anyone know which kits can be OEcombinedı to allow the > analysis of multiple parameters, such as using different fluorochromes from > one kit directly with another kit on the same cells. Thus allowing the > measurement, of say for instance, DNA fragmentation by TUNEL and > phosphatidylserine externalization simultaneously on the same sample. > > My last question (for the day anyway!) is about identifying different > subpopulations of white blood cells (WBCs). What commercial kits, or > fluorochrome conjugates are best, most commonly used, or easiest used. The > type of differentiation I am after is to identify the following > subpopulations: basophils, eosinophils, neutrophils, B lymphocytes, T > lymphocytes, natural killer (NK) cells, and monocytes. Does anyone have > experience doing this? What is the best way? And what works best? > > Many thanks to everyone who reads all my questions, and provides help. > > Mark Hollier. > -- *************************************************************** Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3479 London Research Institute, e_mail: derek.davies@cancer.org.uk Cancer Research UK mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://science.cancerresearchuk.org/sci/facs/ In tenebris lux ***************************************************************Received on Mon Apr 3 12:38:00 2006
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