Be sure to consider the fluorochromes you can utilize in which channels, and chose your viability dye appropriately. In my hands, To-Pro-3 is incredibly bright, and since you have almost no cellular autofluorescence after excitation by the red laser, the difference between positive and negative is huge. But consider this rationale, if you will..... What fluorochrome are you planning to use in FL3? It seems all of the avilable FL3 fluorochromes have at least one knock against them. PerCP is very dim, and though acceptable for highly expressed Ag, like CD8 on T cells, is not very useful on antigen of lesser density. Additionally, there are not many PerCP direct conjugates available, and the streptavidin-PerCP secondary is unreasonably expensive. The tandem conjugates of PE-Cy5 have inconsistent chemistry (between lots, manufacturers, phase of the moon when it was bottled.....), are very photo-labile, and non-specifically bind certin types of cells (monocytes in humans, B cells and APCs in mice, and probably a host of other cell types not yet described. Real fun to trouble shoot!!). And look at what you're losing in FL4. APC is close to the same intensity as PE in our hands. Direct conjugates are widely available, and consistently manufactured. Additionaly, you can buy enough streptavidin-APC secondary to last a year for about $150, allowing you to use the full range of biotinylated antibodies available. Additionally, To-Pro-3 is a bit more expensive, and has less long term stability than other traditional viability dyes. In our hands, after a few months (4-5 if I remember correctly) in the freezer, To-Pro-3 lost almost all its intensity. So while you may be able to make 10 liters of stock To-Pro-3 from one vial, you'll have trouble using it all before it goes bad. So if your experiments revolve around surface antigen identifcation with monoclonal Ab, and exclusion of dead cells, I'd recommend using 7-AAD in FL3. Then you can use the bright APC conjugates in FL4 for your phenotyping. Of couse, you may be using a totally different dye in FL3, in which case this is all moot. Hope this helped someone.... kb -------------------- Keith Bahjat Grad Student (for 10 more months!) University of Florida College of Medicine kbahjat@ufl.edu > 1. We'd like to do some 4-5 color analysis with dead cell exclusion in > FL4. Anybody has any experience with TOPRO-3 and sorts (Mol. Probes)? > How does it compare (sensitivity/specificity) with PI or 7-AAD? > 2. When doing dead cell exclusion, is it necessary to RNAse-treat cells > before applying the DNA-probe (like you would do for cell cycle > analysis); we're working with highly activated cells with high RNA > synthesis and we're concerned about possible uptake of the > nucleotide-probe by mRNA. > > Thanks a lot! > > Karim Vermaelen
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