Hi Heidi! I think that you would have a lot of trouble using PI with a red third colour whether it is a PE-Cy5 tandem (TriColor etc) or PerCP as the emission is wide. 7AAD is an alternative - you could check out this reference: Schmid, I et al Dead cell discrimination with 7AAD in combination with dual color immunofluroescence in single laser flow cytometry Cytometry (1992) 13, 204-208 7AAD emits out in the red so although you will have overlap between PE and 7AAD you should be able to compensate. So you could use DiOc and CD16PE and 7AAD. You dont say which cytometer you would be using because there are alternative strategies. If you have a BD Calibur with the red diode laser you can use TO-PRO-3 (from Molecular Probes) which is excited at 635 and emits at about 660nm; we are routinely using this in three coulor experiments where we also want to remove the dead cells from analysis. If yopu have a sorter equipped with a larger 488nm source you will find that TO-PRO-3 is also excited at 488nm at powers of 50mW or so and so you can use a single laser system. Hope this helps - good luck! Derek On Mon, 11 Jan 1999, Heidi Engelhardt wrote: [ slight editing!] > SO. I need to set up a three colour protocol. I need to label my > targets, label the dead cells, and somehow identify the effector cells > (surface phenotype - they strongly express CD16). Originally I was > considering the following strategy: > > DiOc - to label targets (behaves much like FITC) > PI - to label deads > > and a third label, conjugated to the secondary antibody I use to detect > the CD16-positive cells. > > For this third label, I was thinking about 'Tri-Color' (TC; Caltag). > This combination looked good on paper (DiOc, PI, TC) - the TC > would be excited by the argon laser and emit at a sufficiently high > wavelength to be distinguished from the other two. However, I > discussed this with the Coulter flow cytometry support person, and > she warned me that PI has a very broad emission that interferes > with just about anything other than molecules emitting in the FITC > range. > > She told me about an alternative to PI for live/dead staining - called > 7-aminoactinomycin-D (7-AAD). I have been through the Molecular > Probes catalogue. It seems that 7-AAD is OK for cell cycle > analysis but not so good for viability. Does anyone have any > experience with this? There was a comment in the catalogue > about 7-AAD staining non-differentiated versus differentiated cells. > Staining of viable target cells (tumour cells) would make this stain > useless for our purposes ... (we are ordering the reference for this - > could be that because they were assessing DNA staining rather > than viability, they actually permeabilized the cells first). > > Can anyone suggest alternative strategies? > > Heidi Engelhardt > Department of Animal and Poultry Science > University of Guelph > Guelph, Ontario > CANADA N1G 2W1 > > phone: 519-824-4120, ext. 3658 or 8355 > fax: 519-767-0573 > email: hengel@aps.uoguelph.ca > **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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