Heidi Engelhardt asks about a three-color staining combination for detecting killing of K562 cells by large NK-like cells from the pregnant pig uterus. She writes: >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. > What's "DiOc"? This is half a name for a cyanine dye, where the whole name would be "DiOCn(m), with the n usually a subscript. The n denotes the length of alkyl side chains and the m the length of the polymethine bridge between two heterocyclic rings. DiOC6(3) and DiOC5(3), otherwise called dihexyloxacarbocyanine and dipentyloxacarbocyanine, are dyes which move in and out of cells in response to membrane potential; for permanent labeling you'd probably want n to be 14 or longer, making the dye "lock in" to the cytoplasmic membrane bilayer. Such "tracking dyes" are available from Molecular Probes, among other people. However, a DiO tracking dye will label cells fairly brightly, and the orange tail of its fluorescein-like spectrum will probably make it difficult to detect a phycoerythrin (PE)-labeled antibody. 7-AAD is probably the best choice for live-dead discrimination; it is considerably better for that than for cell cycle (it tends to give high CV's, although it is DNA-specific). This is red (>665 nm, usually "FL3"). PKH26, an indocarbocyanine tracking dye, emits in the same region as PE (about 580 nm, "FL2"). It should be available from Sigma, with the necessary buffers to load cells (there is an art to this). That leaves fluorescein (about 530 nm, "FL1") as a label for CD16; the cells of interest are expressing this strongly, so there shouldn't be a problem detecting them, and the stronger PKH26 and 7-AAD signals, which are at longer wavelengths, should not interfere much. -Howard
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