Hi Saraswathi, I was going to suggest that you use DAPI which we use on the Aria using violet excitation. However as you are using fixed cells most viability dyes are likely to stain all cells (as their membranes will be permeabilised). There are a couple of techniques that you can use to label dead cells prior to fixation - one is ethidium monoazide (which will fluoresce in the same region as your FITC Ab) and the other, which I have used successfully in my Lab, is to use a combination of 7AAD and AD - this was published in the reference below: Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. Schmid I, Hausner MA, Cole SW, Uittenbogaart CH, Giorgi JV, Jamieson BD. J Immunol Methods. 2001 Jan 1;247(1-2):175-86. Again this would mean that you would have to alter your fluorochromes. Can you run the samples unfixed or is that a pre-requisite? If anyone else has any suggestions, I would also be glad to hear them! Best wishes, Derek >Dear Flowers, >I am trying to use a dead cell discriminating dye for an established >Human FACS Panel where surface markers are already conjugated to >FITC, PE, PerCP and APC. I use Becton Dickinson's LSR-II with 3 >lasers : Blue (488nm), Violet (405nm) and Red (633nm). Is there any >dye available that I could exploit using the second (Violet laser at >405nm) or third laser to simultaneously stain and gate out dead >cells in all of my tubes in the Panel. I acquire cells after fixing >with 1%PF. Could you suggest please. Thank you > >Saraswathi Naravula >Scientist II, Dept of Discovery Research >901 California Avenue >Palo Alto, CA 94304-1104 >Ph: 650-496-6550 >Fax: 650-496-1200 >saraswathi.naravula@dnax.org -- *************************************************************** 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 Wed Apr 27 15:38:00 2005
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