Hello Heidi, You didn't mention your laser configuration. If you have a UV laser , you can use Hoechst 33342, or better yet 33258 as a dead/live stain (and apoptotic, for that matter in the case of 33342). I had a poster at ISAC XIX on this .The poster was on using the HO dyes on an arc-lamp system, but the protocol is the same for a UV-laser equipped cytometer. See: Lopez,PA, Ottenberg,MJ. Rapid cellular viability quantification using an arc-lamp flow cytometer. Cytometry 1998; Suppl.9:143 Peter > ---------- > From: Heidi Engelhardt[SMTP:hengel@APS.UoGuelph.CA] > Sent: Monday, January 11, 1999 10:10 AM > To: Cytometry Mailing List > Subject: (Fwd) 'alternative' live/dead stains? > > > Hello! I would like to pick the brains of the group for help in > setting > up a three colour protocol. So far, my experience is > limited to double staining, using FITC and PE. I have had no > official training on the flow cyt, so there are huge gaps in my > knowledge ... > > I am trying to set up a flow cytometric assay to detect lysis and > conjugate formation between target cells (K562 myeloid leukemia > cell line) and effector cells (natural killer (NK)-like lymphocytes > from the pregnant pig uterus). Uterine cells will be a mixed > population, with only a proportion of them lytic. > > Another complication is that the uterine lymphocytes likely to be > mediating the killing are much larger than conventional NK cells. > Normally, one can distinguish targets from circulating NK cells by > size, but I suspect that my uterine effectors will be similar to the > target cells in size and granularity. > > 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? > > Thanks! > > Heidi Engelhardt > > > 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 >
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