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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