Heidi, Comments and suggestions, listed where relevant . . . Heidi Engelhardt wrote: > 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. . . . > I don't know in the pig, but we did similar work in mice, analyzing T cells. I found these lymphs to be similar in size and 90 scatter to peripheral and spleen associated lymphs, so I wouldn't assume light scatter will be useless. You can use peripheral lymphs to help you target the uterine lymphs in the complex light scatter distribution you're likely to see. > 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) Looking at the emission spectrum of DiOC6, it emits optimally around 500nm, well below most FITC (FL1) bandpass filter limits . . . I've found its signal to be somewhat dim, so alternatives might be advantageous. It does work well with PI, though. > PI - to label deads . . . > You might also consider CFSE for the target cell label (see <http://jcsmr.anu.edu.au/facslab/CFSE1.html>). We've used CFSE to follow proliferation cultures for weeks . . . works well combined with surface antigen labels. BCEFC is also a long lasting label for tracking cells (see <http://www.probes.com/handbook/ch16-3.html>). Or, alternatively, you could use a human CD45 to differentiate targets from the pig effectors, but that's a different direction . . . > . . . 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. > We routinely use PI as a viability marker in FITC/PE labeled samples . . . works well. You do need to reduce the PI concentration so it doesn't interfere -- about 1ug/ml final works well. Also, instead of looking for the PI around 630, use a 675 bandpass filter (FL3 on a Scan or Caliber, FL4 on the XL). Actually, I think you'll have more problems separating PI from TC, due to the relative signal strengths. > 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? . . . 7-AAD will work as well . . . I believe there are examples in the literature of applications using 7-AAD this way. > 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). Yes. > Can anyone suggest alternative strategies? > I'd start with the DIOC6, combined with your CD16-PE and PI for viability. If it doesn't work, then think of alternatives.This lives and dies by your controls, however, so give lots of thought to both single color and assay controls. Good luck. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry kukuruga@medmail.med.umich.edu
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